Synthesis of recombinant antibacterial proteins in the ...

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Synthesisofrecombinantantibacterial

proteinsintheChlamydomonasreinhardtii

chloroplast

MaximilianEdwardAlfredBlanshard

UCL

AthesissubmittedforthedegreeofDoctorofPhilosophy

September2017

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Declaration

I,MaximilianE.A.Blanshard,confirmthattheworkpresentedinthisthesisismy

own.Whereinformationhasbeenderivedfromothersources,Iconfirmthatthis

hasbeenindicatedinthethesis.

…………………………………………………………………

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Acknowledgments

Firstly,Iwouldliketothankmysupervisor,ProfSaulPurton,fortheopportunityto

studyinhislabforthelastfouryears,andforprovidinghelp,encouragement,and

ideassofreelyandwillingly.Hugethankstomysecondarysupervisor,DrElaineAllan,

whosesupportandkindwords,aswellastimeinhelpingmetocompletemilestones,

are hugely appreciated. Thank you also tomy thesis committee chair,Dr Richard

Hayward, who’s ability to constructively criticise and encourage a methodical

approachwasinvaluable.DrMelindaMayer,mycollaborator,deserveshugethanks

foragreeingtohelpoutwiththeCD27Lworkandrunningsomanyturbidityreduction

assaysforme.

Iwould like to thank allmembersof thePurton Lab, past andpresent. From the

beginningtotheendofmyPhD,DrLauraStoffelshastaughtmehowtobeascientist

andheramazingabilitytoknoweverything(andwhereeverythingis!)neverceases

toamazeme,soIwouldliketothankherforallofthehoursoftimeandfrustration

shehasputintohelpingme,andresistingtheurgetosay“Justlookitup”.Iwould

alsoliketothankDrRosieYoungforteachingmesomuchmolecularbiologyandfor

alloftheencouragementwhenexperimentsfailed.Anenormousthankyoutoour

labmanager,ThushiSivagnanam,withoutwhomItrulybelievenothingwouldever

getdone–yourworkisremarkableandwedonotsayitoftenenough.Thankyouto

DrHenryTaunt,whoestablishedtheendolysinworkandhasplayedan important

part inmyPhDdespitesupposedly leavingthe lab in2013, includingbuyingmore

thanhisfairshareofroundsandgivinginsightintotheworldofhome-brewing.

TomyfriendsandcolleaguesinthePurtonlabwithwhomIhavemoaned,celebrated,

travelled, andgenerallyhad fun– I couldnothavedone itwithout you.Dr Janet

Waterhouse,DrAliceLui,DrPriscillaRajakumar,andDrUmaimaAlHoquanimade

mefeelwelcomefromthedayIarrived.ToJulianadeCostaRamos,XeniaSpencer-

Milne, Fiona Li, SaowalakChankgoandMarco LarreaAlvarez thank you forbeing

greatfriendsandbestofluckwiththerestofyourPhDs.

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Mostimportantly,Iwouldliketothankmyfamily,inparticularmyMumandDadfor

theirexceptionalgenerosityandunbelievablekindness throughoutmy life,and in

theirpatiencewithallofmylifedecisions.Iamsoluckytohavethecoolestparents

intheworld.Icouldnothavedoneitwithoutyou!

Last,andmostcertainlynotleast,IwouldliketothankSamanthaHunt,whoselove,

companionship and supportmade even themost frustratingmoments in the lab

bearable,andwhohasfilledthelastsevenyearswithadventuresandfun.Thankyou!

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Abstract

Theriseofantibioticresistanceandthedeclineinantibioticdiscoveryhavebeenwell

publicised. These issues, in combination with a growing global reliance upon

antibiotics for everyday modern life, urgently require the discovery of novel

antibacterialdrugs.

Endolysins are one potential candidate to support, or replace, conventional

antibiotics. Endolysins are lytic enzymes produced by bacteriophage in natura to

enablethereleaseofviralprogenyfrominsidethehostbacterium.Whenapplied

exogenously,endolysinscanlyseGram-positivebacteria,andthuscouldbeusedas

anovelantibacterialforthisgroupofpathogens.

Biologicallyactiveendolysinshavebeensuccessfullyexpressedasrecombinants in

thechloroplastofthegreenalga,Chlamydomonasreinhardtii.C.reinhardtii,andin

particular the chloroplast, has several features as a cell factorywhichmake it an

attractivealternativetothetraditionalrecombinantproteinproductionplatforms.C.

reinhardtii isfreeofendotoxins,canbecultivatedatlowcostinphotobioreactors,

hasGRASstatus,andisgeneticallytractable.

This study initially focuses upon improving the accumulation and activity of one

endolysin,Cpl-1,targetingStreptococcuspneumoniae.RecombinantCpl-1hasbeen

shown previously in the Purton lab to accumulate to moderate levels in the C.

reinhardtii chloroplast.Herewepresent two transgenic linesofC. reinhardtii that

appear to accumulate recombinant Cpl-1 to higher levels – one through the

incorporation of multiple expression cassettes, and one through codon pair

optimization. To improve the activity of Cpl-1 as an enzyme, Cpl-1 binding site

mutagenesis,Cpl-1dimerization,andtheproductionofapotentiallysynergisticholin

proteinwereallattempted.

Finally,anendolysinagainstClostridiumdifficile,CD27L,wassuccessfullyproduced

intheC.reinhardtiichloroplastandshowntobeactiveinvitro.Anotherendolysin,

thistimetargetingPropionibacteriumacnes, failedtoexpress inC.reinhardtii,but

wasexpressedinE.coli,albeitwithoutobviouslyticactivity.

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Abbreviations

aadA aminoglycoside-3’’-adenyl-transferasegene

Amp ampicillin

AMPS ammoniumpersulphate

BHI brainheartinfusion

BME β-mercaptoethanol

bp basepairs

BSA bovineserumalbumin

CBD cellbindingdomain

CD catalyticdomain

CES controlbyepistasisofsynthesis

CFU colonyformingunit

CPK Corey,Pauling,Koltun

CTB choleratoxinB

DNA deoxyribonucleicacid

dNTP 2’deoxynucleoside5’-triphosphate

EDTA ethylenediaminetetraaceticacid(disodiumsalt)

e.g. exempligratia=forexample

ELISA enzyme-linkedimmunosorbentassay

g gram

GC guanineandcytosine

GFP greenfluorescentprotein

GOI geneofinterest

HA hemagglutinin

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His histidine

HSM highsaltminimalmedium

IPTG Isopropylβ-D-1-thiogalactopyranoside

kb kilobasepairs

kDa kiloDalton

LB luria-bertanimedium

mg milligram

min minute

ml millilitre

mM milimolar

mRNA messengerribonucleicacid

mt mating-type

NCBI NationalCentreforBiotechnologyInformation

OD opticaldensity

ORF openreadingframe

PCR polymerasechainreaction

PDB proteindatabank

PEG polyethyleneglycol

PG peptidoglycan

PMF protonmotiveforce

PSI photosystemI

PSII photosystemII

psi poundspersquareinch

RCSB ResearchCollaboratoryforStructuralBioinformatics

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RNA ribonucleicacid

rRNA ribosomalribonucleicacid

SD Shine-Dalgarnoconsensussequence

SDS sodiumdodecylsulphate

SDS-PAGE sodiumdodecylsulphatepolyacrylamidegelelectrophoresis

STGG skimmedmilktryptoneglycerolglucosemedium

Strep streptavidin

TAP trisacetatephosphatemedium

TBS trisbufferedsaline

TBS-T trisbufferedsaline–tween20

TCP totalcellprotein

TEMED N,N,N’,N’-tetramethylethylenediamine

tris tris(hydroxymethyl)aminomethane

TRA turbidityreductionassay

tRNA transferribonucleicacid

TSP totalsolubleprotein

TSY trypticasesoyyeastextractmedium

UTR untranslatedregion

v/v volumeforvolume

w/v weightforvolume

WGS wholegenomesequencing

WHO WorldHealthOrganisation

WT wildtype

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CHAPTER1INTRODUCTION 15

1.1 Anewgenerationofantibiotics 15

1.1.1 Whyareantibioticssoimportant? 15

1.1.2 Whydoweneednewantibiotics? 19

1.1.3 Whatalternativestoantibioticscurrentlyexist? 24

1.2 Anoverviewofbacteriophageendolysins 28

1.2.1 Thebiologicalroleofbacteriophageendolysins 28

1.2.2 Thetherapeuticpotentialofendolysinsand“exolysis” 32

1.2.3 Otherapplications 38

1.3 Producingrecombinanttherapeuticproteins 39

1.3.1 Commonexpressionplatforms 39

1.3.2 Microalgaeasexpressionplatforms 41

1.3.3 Chlamydomonasreinhardtiiasanexpressionplatform 44

1.4 Targets/applications 51

1.4.1 Streptococcuspneumoniae 51

1.4.2 Clostridiumdifficile 53

1.4.3 Propionibacteriumacnes 54

1.5 Summary,aimsandobjectives 55

CHAPTER2 MATERIALSANDMETHODS 57

2.1 Strains,media,cultureconditionsandcelldensityquantification 57

2.1.1 Chlamydomonasreinhardtii 57

2.1.2 Escherichiacoli 63

2.1.3 Streptococcuspneumoniae 64

2.1.4 Clostridiumdifficile 65

2.1.5 Propionibacteriumacnes 66

2.2 Plasmids 67

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2.3 Molecularbiology 69

2.3.1 Buffers 69

2.3.2 Genedesignandsynthesis 70

2.3.3 Polymerasechainreaction(PCR) 70

2.3.4 PCRDNAPurification 71

2.3.5 AgarosegelElectrophoresis 71

2.3.6 DNAgelextraction 71

2.3.7 SitedirectedmutagenesisbyPCR 72

2.3.8 DephosphorylationusingAntarcticPhosphatase 73

2.3.9 Blunt-endDNAcloning 73

2.3.10 DNARestrictionendonucleasedigestion 73

2.3.11 DNALigation 73

2.3.12 CompetentE.colitransformation–Heatshock 73

2.3.13 C.reinhardtiitransformation–glassbead 74

2.3.14 E.colicolonyPCR 75

2.3.15 E.coliplasmidisolation–CrudeandKit 75

2.3.16 E.colitestdigest 75

2.3.17 C.reinhardtiigenomicDNAisolation 75

2.3.18 DNASequencing 76

2.4 Proteinanalysis 76

2.4.1 Preparationoftotalproteinextracts 76

2.4.2 Sodiumdodecylsulphatepolyacrylamidegels(SDS-PAGE)� 77

2.4.3 Proteinpreparationfordotblot 78

2.4.4 CoomassieBrilliantBlueRstaining 78

2.4.5 Westernblotanalysis 79

2.4.6 Immuno-detection 79

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2.4.7 Proteinquantificationassay 80

2.5 C.reinhardtiiproteinpurification 80

2.5.1 Crudeextractpreparation 80

2.5.2 Ammoniumsulphateprecipitation 81

2.5.3 Anti-StrepIIpurificationcolumn 81

2.6 E.coliproteinpreparation 81

2.6.1 IPTGinduction 81

2.6.2 BugBuster™(Novagen) 82

2.6.3 HighPressureHomogenizer 82

2.6.4 Sonication 82

2.6.5 Disulphidebondformationinvitro 83

2.7 Antibacterialactivityassays 83

2.7.1 Colonyformingunitassay 83

2.7.2 Turbidityreductionassay 84

CHAPTER3 IMPROVINGACCUMULATIONLEVELSOFCPL-1INTHECHLOROPLASTOFCHLAMYDOMONASREINHARDTII 86

3.1 Introduction 86

3.1.1 IntroductiontobacteriophagelysinCpl-1 86

3.1.2 Factorstoconsiderinproteinaccumulation 88

3.1.3 EpistasyofsynthesisintheC.reinhardtiichloroplast 89

3.1.4 CodonusageintheC.reinhardtiichloroplastgenome 90

3.2 Aimsandobjectives 90

3.3 Resultsanddiscussion 91

3.3.1 Investigationintomeasuringproteinaccumulationlevels 91

3.3.2 Investigating control by epistasy of synthesis in proteinaccumulation 105

3.3.3 Codonpairoptimizationforimprovementofproteinaccumulationlevels 124

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3.4 Conclusionandfuturework 140

3.4.1 Multipleexpressioncassettestrategy 140

3.4.2 Identicaltransformanttransgenevariability 142

3.4.3 Codonpairoptimisation 144

CHAPTER4 IMPROVING THE ACTIVITY OF CPL-1 AGAINST STREPTOCOCCUSPNEUMONIAE 146

4.1 Introduction 146

4.1.1 ManipulatingCpl-1bindingactivity 146

4.1.2 Dimerisationasamethodtoimproveproteinstability 147

4.1.3 Theroleofholinsinthebacteriophagelyticcycle 150



4.3.1 ManipulationoftheCpl-1cholinebindingsitestoimproveactivity 153

4.3.2 ImprovingCpl-1stabilitythroughdimerisation 176

4.3.3 ExpressionoftheCpl-1associatedholin,Cph-1 186


4.4.1 Cellwallbindingsitemutagenesis 193

4.4.2 Cpl-1dimerisation 196

4.4.3 Holinproduction 197

CHAPTER5 EXTENDING THE CHLAMYDOMONAS REINHARDTII CHLOROPLASTPLATFORMTOOTHERENDOLYSINS 200

5.1 Introduction 200

5.1.1 PreviousendolysinexpressionintheC.reinhardtiichloroplast 200

5.1.2 Theneedformoreendolysins 201



5.3.1 Clostridiumdifficile,ΦCD27andCD27L1-179 203

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5.3.2 C.difficileendolysinCD27L 203

5.3.3 Propionibacterium acnes, bacteriophage PA6 and its endolysinGp20 218


5.4.1 CD27Lendolysin 228

5.4.2 Gp20endolysin 232

5.4.3 Futureendolysins 238

CHAPTER6 FINALDISCUSSIONANDFUTUREPROSPECTS 240

6.1 Summary 240

6.1.1 Summaryofresults 240

6.1.2 Summaryofshort-termfuturework 241

6.2 Discussionandlong-termfutureprospects 243

6.2.1 Alternative strategies to improving expression levels in C.reinhardtiichloroplast 243

6.2.2 Othermicroalgalhostsforscale-up 246

6.3 Concludingremarks 248

6.3.1 Viewsonthefutureofantibiotics 248

6.3.2 Viewsonthefutureofmicroalgae-producedtherapeutics 249

REFERENCES 250

CHAPTER7 APPENDICES 274

7.1 ResultsChapter3Appendices 274

7.1.1 AppendixA 274

7.1.2 AppendixB 275

7.1.3 AppendixC 276

7.1.4 AppendixD 277

7.1.5 AppendixE 277

7.1.6 AppendixF 278

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7.1.7 AppendixG 280


7.2.1 AppendixH 284

7.2.2 AppendixI 285


7.3.1 AppendixJ 286

7.3.2 AppendixK 287

7.3.3 AppendixL 287

7.3.4 AppendixM 288

7.3.5 AppendixN 289

7.3.6 AppendixO:ListofPrimers 289

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Chapter1 Introduction

1.1 Anewgenerationofantibiotics

1.1.1 Whyareantibioticssoimportant?

1.1.1.1 History

SirAlexanderFleming(1881-1955)isbestknownforhisdiscoveryofbenzylpenicillin

(PenicillinG)in1928,anobservationthatshapedthemodernworldandopenedup

hugevistasofmodernmedicalandsurgical techniques thathavebecomeroutine

across the globe. After the initial discovery of penicillin by Fleming at St Mary’s

Hospital in London, it took a further 16 years of refinement, optimization and

collaborationbetweenindustryandacademiabeforepenicillincouldbeproducedin

sufficientvolumes to treathumans (Zaffirietal.2012). Incontrast to thepopular

storyofFlemingstumblingacrosspenicillinonhisreturnfromholiday,thescalingup

ofproductionwasnomeanfeat.After12yearsofpromotingpenicillinandsearching

forsolutionstothepurificationandstabilityissuespenicillinfaced,Fleminghadall

butgivenuponhisdiscovery(Aminov2010).However,duetothepressuresofthe

secondworldwar,theworkwascontinued,thoughstillitdidnotcomeeasy:asJohn

L.SmithofPfizersaidatthetime,“Themouldisastemperamentalasanoperasinger,

theyieldsarelow,theisolationisdifficult,theextractionismurder,thepurification

invitesdisaster,and theassay isunsatisfactory.” (Werth2014).By1945however,

massproductionanddistributionofpenicillinhadbeenachieved–theworld’sfirst

commerciallyavailableantibiotic.ThechemicalstructureofPenicillinGanditsmode

ofactionasdisplayedinFigure1.1.

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FigureRemoved

Figure1.1Thesmallmoleculebenzylpenicillin(PenicillinG),knownasaβ-lactamduetothepresenceofaβ-lactamringinitsmolecularstructure(Drugbank2017).Aswithmostβ-lactamantibiotics, benzylpenicillin functions throughdisrupting cellwall biosynthesis inbacteria.The image on the right shows benzylpenicilin inhibiting formation of crosslinks in thebacterial peptidoglycan cell wall. Normally transpeptidases (also called penicilin-bindingproteins) bind to D-alanyl-D-alanine pentapeptide of the peptidoglycan monomers andcatalyse cross-linking via a peptide bridge. However, penicillin resembles the D-alanyl-D-alaninepentapeptideandblockstheactivesiteofthetranspeptidases,preventingmonomerpolymerisation.Thelackofcross-linkingweakensthecellwall,causingosmoticlysis.ImagereproducedfromKaiser(2011).

PenicillinGwasthefirstofmorethan20distinctantibioticclassestobediscovered

fromthe1940stothe1960s,inwhatisnowrecognisedasagoldeneraofantibiotic

discovery (Arias andMurray 2015). These smallmoleculeswere all isolated from

bacteria and fungi for their ability to block key enzymatic steps in bacteria, and

presumablyservetogivemicroorganismsacompetitiveadvantageinenvironmental

niches(Lewis2013).However,therearealsotheoriesthatthesesmallmoleculesact

ascell-cellcommunicationsystemsatlowconcentrations,giventheirvastdiversity

andtheirabilitytomodulatecelltranscripts(Davies2006).Regardlessoftheirnatural

role,theseidentified antibiotics targeted several different aspects of bacterial

machineryessentialforcellmaintenanceandreproduction,anillustrationofwhich

ispresented inFigure1.2.The incredibleefficacyofantibiotics, theirrapidrateof

discoveryandtheirabilitytocurepreviouslydeadlyinfectionsinamatterofdaysled

tothembeinghailedasa“magicbullet”(Senguptaetal.2013).Theywerenotonly

valuableascurestobacterialinfections,butinpreventiontoo,thusopeningupvast

avenuesformedicineandindustrywhichwillbefurtherexploredinthenextsection.

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FigureRemoved

Figure1.2Thevariousmodesofactionofdifferentantibioticclassestokillbacteria.Itshouldbe noted that many classes share the same targets, for example cell-wall synthesis isinhibitedbyfiveseparateclassesofantibiotics.ReproducedfromLewis(2013).

1.1.1.2 Applications

Themostobviousapplicationforantibioticsisintreatingbacterialinfections.When

penicillinwas firstmass produced,WorldWar IIwas still raging and stockswere

earmarkedforsoldiersonthefrontline,sufferingfromstaphylococcal,streptococcal,

andpneumococcalwoundinfections(MailerandMason2001).Whileinfectionsof

minorcutsandinjuriescanstillwarrantantibioticsonoccasion,betterknowledgeof

hygiene,vaccinations,andafter-carehavereducedthismarket.

Prophylactic antibiotics on the other hand are still of immense importance and

enableawholesuiteofsurgeriesandchemotherapieswhichmanytakeforgranted.

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Allinvasivesurgeries,fromorgantransplantstodentalwork,areaccompaniedwith

aprophylacticcourseofantibiotics.Immunosuppressingchemotherapiesforcancer

treatmentsrelyonantibioticstostopbacterialinfectionswhilethebody’simmune

systemisoutofaction(O’Neilletal.2015).Inshort,accesstoantibioticsunderpins

vastnumbersofsurgeriesandmedicaltechniquesusedinmodernmedicine.

Oneofthemostcontroversialusesofantibioticsisasgrowthpromotersinlivestock.

This stems from the discovery that sub-therapeutic levels of antibiotic cause

increasedgrowth.Themechanismforthisremainsunclear,butthemostlikelytheory

seems to be that growth is more efficient without gut bacteria competing for

nutrients(Gaskinsetal.2002).Notonlydolowlevelsofantibioticsimprovegrowth

rates, but also conception rates and feed conversion ratios, altogether having a

pronouncedimpactuponglobalmeatproduction(Haoetal.2014).

Veterinarymedicine is another large consumerof antibiotics, enabling farmers to

rearanimalsincloserproximitytooneanothersuchasinbarnswherepreviouslya

build-upofpotentiallyinfectiousbacteriawouldpreventhighanimaldensitiesand

continuousfarming.Economically,thishasbeenadvantageous–increasingfarming

efficienciesandloweringfoodprices.Thesameistrueoffishfarming:thegrowthof

aquaculture to industrial scales has been almostwholly reliant uponprophylactic

antibioticuse,enablingthehighdensityfarmingoffishtomeetthegrowingglobal

demand(Cabello2006).

The remarkable reliance that the modern world has built upon antibiotic

consumption leads us to worry about what would happen if they ceased to be

effective.Wehavementionedbrieflyjustafewofthehugeindustriesthatuse,and

over-use,antibiotics.Aswewillseeinthefollowingsection,antibioticsareafinite

resourceduetotheemergenceofresistantbacteriaand if these industriesareto

continuetogrow,thennewantibioticsmustbesought.

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1.1.2 Whydoweneednewantibiotics?

1.1.2.1 Resistance

TheWorldHealthOrganisation(WHO)definesantibioticresistanceasachangewhich

occurstobacteriawhenexposedtoantibiotics,resultingintheantibioticbecoming

ineffective and the persistence of the infection (WHO 2016). This definition is

valuableatatherapeuticlevel,butthissectionwillgiveanoverviewofhowantibiotic

resistancefunctionsamolecularlevel.

ThefivemechanismsofantibioticactionillustratedinFigure1.2representtheentire

variability within our antibiotic arsenal. Given how important antibiotics are to

modernsociety,thisisanalarminglysmallnumberofbacterialtargetstorelyupon,

giventhatthereareknowntobeapproximately200conservedessentialbacterial

proteins(Lewis2013).Resistancetoeachofthesetargetshasarisenquicklyaseach

newdrughasbeenusedonalargescale,withresistancetomorerecentantibiotics

suchasLevofloxacin(asecondgenerationfluoroquinolone)beingreportedin1996,

the same year as it was introduced (Ventola 2015). It is therefore important to

understand how resistance is acquired both at the molecular level, so that new

therapiesanddrugcombinationscanbeproduced,andatthepopulationlevel,so

thatpreventativemeasuresandprocedurescanbeimplemented.

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FigureRemoved

Figure1.3Thesixmajorantibioticresistancemechanisms.Resistancemechanismsarenotnecessarilyspecific toacertainclassofantibiotics,withmanyantibioticsbeingsubjecttomultipleresistancemechanisms.Sometherapiestargettheresistancemechanisminordertorestoreantibioticefficacy,forexampletheapplicationofβ-lactamaseinhibitorsalongsideβ-lactamantibiotics.ReproducedfromLewis(2013).

Resistancearisesduetothehighselectivepressurethatantibioticsputuponbacterial

populations.Eachmodeofresistancetoeachofthefivesitesofantibacterialaction

isillustratedinFigure1.3.Forexample,penicillinisaβ-lactam,aclassofantibiotics

that inhibit cell wall synthesis in bacteria. β-lactams bind to penicillin-binding

proteins(PBPs),whichareresponsibleforbacterialcellwallcross-linking,andinhibit

theiraction.Theresultinginabilityofthebacteriatocorrectlyformacellwallleads

toautolysis(Fernandesetal.2013).Severalmethodsofresistancehavedeveloped

towards β-lactams. Firstly, target modification: modification of PBPs to exhibit a

muchloweraffinitytopenicillin.Secondistheproductionofβ-lactamases,enzymes

whichdegradeormodifyβ-lactams,andthirdlyarechangestopermeabilityinthe

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cellwalltoβ-lactams,whichcaneitherblockentryoractivelyremovetheβ-lactams

via efflux proteins (Fernandes et al. 2013). In this single example three different

resistancemechanismsexistforasingleantibiotic,highlightingtheremarkableability

ofbacteriatoevolveunderantibioticinducedselectivepressureandunderliningthe

realpotentialforsomeantibioticstobecomeobsolete.

Itisreasonabletoaskhowantibioticresistanceevolvesandspreadssorapidly.Two

categories for resistance can be formed: endogenous resistance and exogenous

resistance. Endogenous resistance refers to resistance arising within the bacteria

itself i.e. bymutationand selection. Exogenous resistance involves thepassingof

resistance genes between different bacterial species via horizontal gene transfer

(Silver2011),eachofwhichisexploredbelow.

Endogenousresistanceisoftengeneticallyrecessiveasittendstoresultinalossof

function, for example reduced permeability to antibiotics, which have other

(negative)consequencesfornormalcelloperation.Ifantibioticselectioniscontinued

foralongtime,thensecondarymutationsmayeventuallyevolvetocompensatefor

lossoffunction.However,generallythesemutationswilleventuallybelostafterthe

removalofselectivepressure.

Exogenously acquired resistance on the other hand generally involves a gain of

functionandisthereforemorestable,forexampletheabilitytosynthesizeantibiotic-

degradingenzymes.Apartfromaslightmetabolicburden,thesegainsoffunctionare

not generally detrimental to the bacterium. Exogenous resistance is therefore of

mostconcernclinically,asresistancepersistsafterantibiotictreatmenthasfinished

(SilverandBostian1993).

Themanyusesofantibioticsdescribedin1.1.1.2provideperfectopportunitiesfor

resistance to be selected. For example, the lack of distinction between “human

antibiotics” and “veterinary antibiotics” has dangerous implications for emerging

resistanceviahorizontalgenetransfer.Furthermore,themannerinwhichantibiotics

aregenerallydeliveredtolivestock,suchasintheirfoodorwater,canleadtosub-

inhibitory concentrations of antibiotic which provides an ideal environment for

resistanceselection(Meeketal.2015).

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FigureRemoved

1.1.2.2 Antibioticdevelopment

Duringthegoldeneraofantibioticdiscovery,arangeofbroad-spectrumantibiotics

werequickly identified,patentedandscaled toproduction,but today thesesame

chemicalsstillformthebasisofourantibioticarsenal.Fromthemid-1960sonwards,

onlysixnew,approved,antibioticclasseshavebeendiscovered,andnonesincethe

discoveryofdaptomycinin1986.

Figure1.4The“GoldenEra”ofantibioticdiscoveryproducedthebasisforalloftheantibioticsuponwhichwestill rely today.Modificationsandsyntheticvariantsof theseclasseshaveextendedthelifeofmanyofthesedrugs,butalackoftrulynovelantibioticspersists.Veryrecentlynewbacterialculturingtechniqueshaveraisedhopesofnewantibioticclassesbeingdiscoveredandapproved(e.g.teixobactin),andarediscussedlaterinthissection(Lingetal.2015).ImagereproducedfromSilver(2011).

Thisalarmingdrop-off inantibioticdiscovery isduetoseveral factors. Intheearly

daysofantibioticresearch,discoverywaslargelythroughempiricalscreening–this

amounted to testing of various microorganism extracts for bacterial growth

inhibition, requiring little understanding of their action. This method worked

23

extremelywellinthe1940s,50sand60s,butsoonthese“lowhangingfruits”hadall

been discovered. These methods soon led to replication of discovery; common

antibiotics were being repeatedly discovered, thus reducing the efficiency of the

discoverysystem.Themethoddeployedshiftedtowardstarget-directedscreensand

eventually big pharma companies reallocated resources to more lucrative

therapeutics(Livermore2011;Silver2011).Economically,theantibioticmarkethas

been somewhat a victim of its own success: withmany competing drugs on the

market, the price pharmaceutical companies can charge per prescription of

antibioticsismuchlowerthanthatofmanyotherdrugs(Bax1997).

However, the news is not all bad. Although no (or few) new classes have been

discovered since the 1980s, there have been improvements within the existing

classes.Whilesuchmodificationsdoincreasetheefficacyoftheseantibioticsfora

limited period of time, the fundamental resistance mechanisms still exist and

resistanceoftenquicklyre-emerges(Payneetal.2007).Othertacticshaveinvolved

blockingtheresistancemechanisms,suchasthroughuseofβ-lactamaseinhibitors

(WalshandWencewicz2014),anddeliveringtheseincombinationwithexistingβ-

lactam antibiotics. But none of these techniques broaden the limited number of

targetsthatourantibioticarsenalcurrentlypossessesandhavebeenincrementalin

nature.

New strategies to specifically target virulence factors, thus applying less selective

pressureandwithresistancebeingatthecostofvirulence,presentneatalternative

targetsforantibiotics,butremainalongwayfromtheclinic(Clatworthyetal.2007).

Combinationsofantibioticsarenowthenorm,andcanachievesynergisticeffects.In

somecases,resistancetooneantibioticmayincreasesusceptibilitytoanother–for

example colistin acts as a highly synergistic combination with several other

antibiotics,byimprovingthepermeabilityofthebacterialoutermembraneinGram

negativebacteria(Tängdénetal.2014).

Oneof thegreatest restraints to the“GoldenEra”ofantibioticdiscoverywas the

limitednumberofmicrobesthatcouldbegrowninthelaboratory,andthentested

for antimicrobial properties. In fact, it is estimated that 99% of bacteria are

24

“uncultured”andthusuntapped.Thedevelopmentofnewtechniquesforculturing

soilmicrobesinsituenablestheproductsofthesemicroorganismstobestudiedfor

the first time. This technology has already produced a new antibiotic candidate

teixobactinwhichbindslipidIIandIII,therespectiveprecursorsofpeptidoglycanand

cellwallteichoicacid,andthusinhibitscellwallsynthesis(Lingetal.2015).

Itisclearthat,despiterecentinnovations,thedearthofrecentantibioticdiscovery

combinedwiththeriseofantibioticresistanceisaworryingstateofaffairsforour

modernwayoflife.Bettercontrolofcurrentantibioticusagemayslowtherateof

resistanceoccurrence,butnewantibioticsaredesperatelyneededtocompeteinour

evolutionaryarmsraceagainstbacteria.

1.1.3 Whatalternativestoantibioticscurrentlyexist?

Thearmsraceagainstbacteriawillneverend,andevenwhennewantibioticswith

newtargetsarediscovered,resistancewilleventuallyarise.Itisthereforevitalthata

robustpipelineforantibioticproductionisinplace,andgiventhelackindiscoveryof

conventionalantibioticsoverthepast50years, itmakessensetolooktoplugthe

gaps with alternative antimicrobials. With the advancement of knowledge and

technologyinthesenovelantibioticfields,itislikelythateventuallyone,ormany,of

these“alternativeantibiotics”willbecomethenew“conventionalantibiotics”.

1.1.3.1 Non-lethaltreatments

Whilenotstrictly“antibiotics”,non-bactericidaltherapiesdeserveabriefmentionin

this literature review as they provide an alternative treatment to traditional

antibiotics.Theyalsoopenupadifferentwayofthinkingaboutbacterialinfections,

namelythatdamagetohostorganismswhensufferingfrombacterialinfectioncan

becausedasmuchbyhostresponseasbymicrobialfactors(PirofskiandCasadevall

2008). Efforts to prevent the host response, through tactics such as anti-

inflammatorymonoclonalantibodiescanpotentiallyminimisehostdamagewhilethe

body’simmunesystemfightstheinfection.

Othermethodsinvolvehinderingtheactivityofthebacteriathroughcompetitionfor

resources, either by the introduction of other bacteria (probiotics) or the

25

sequestration of host nutrients (Spellberg 2014). Furthermore, bacterial toxin

inhibitorsandbiofilminhibitorswhichdonotdirectlyharmthebacteria,butreduce

theirpathogenicityofferapromisingalternativepath.Amajoradvantageofthese,

alongwithhost-responsemodulation, is that theydonotapply the same levelof

selective pressure on the bacteria in question to evolve resistance as traditional

bactericidalmeasures(Chengetal.2014).

1.1.3.2 Predatorybacteria

Somebacteria,suchasBdellovibrio,PeredibacterandVampirococcus,preyonother

bacteria and therefore may offer a mechanism for active control of bacterial

infections.Thesebacteriagoonestep further thancommensal ‘probiotics’,which

generally compete for nutrients and resources thus reducing the scope for

opportunistic pathogenic bacterial colonisation, by entering host bacteria and

causing cell lysis (Dwidar et al. 2012). Predatory bacteria, widely known as

Bdellovibrio-and-like-organisms (BALOs), have demonstrated killing of pathogenic

bacteriainvitro(VanEsscheetal.2011),andrecentinvivostudiesshowareduction

inbacterialburdenofKlebsiellapneumoniaeinratlungs(Shatzkesetal.2016).

AninterestingstrengthofBALOsistheirabilitytopreyuponGram-negativebacteria,

whichareamajorsourceofglobaldisease.Theouter-membraneofGram-negative

bacteria presents a barrierwhich severalmajor antibiotics (e.g. vancomycin) and

antibiotic-alternatives(e.g.endolysins)struggletopenetrate(Miller2016).Another

advantageisthatpreyresistanceisextremelyrare,withonlyonecasesofarreported

(Varon1979),suggestingaderivedtherapycouldbeeffectiveforasubstantialperiod

oftime.

However, safety concerns around inoculating humans with bacteria must be

addressed. A potentially toxic response to the lipopolysaccharides in the Gram-

negative outer-membrane of BALOs is yet to be solved. Furthermore, predatory

bacteriaareunabletoeradicatealltheirprey,evenwhenappliedinveryhighratios

(SockettandLambert2004),andarealsostrictlyaerobicthuslimitingtheirpotential

for internalhumanapplication.Theymayhowever findaplace in therapieswhen

26

usedtogetherwith,forexample,β-lactamstowhichtheyaretolerant(Dwidaretal.

2012).

1.1.3.3 Antimicrobialpeptidesandbacteriocins

Antimicrobialpeptides(AMPs)aresmallpeptides(10-50aminoacids)thatformpart

oftheinnateimmunesystem,andarefoundinallclassesoflife.AMPshavebeen

shown to act independently as antimicrobials, but also to have wide ranging

immunomodulatory functions in mammals, including altering gene expression,

promotingwound healing andmodulating dendritic cell response (Bowdish et al.

2005;Fernebro2011).Furthermore,AMPscandisplayactivityagainstvirusesand

fungitoo,thusearningtheuseoftheword“antimicrobial”,ratherthan itssubset

“antibiotic”.AMPsalsohaveactivityagainstbiofilms,whichcanharbourbacteriain

chronicdiseases andare thereforean interesting routeof research. Themodeof

actionofAMPs is stillunclear,although it isbelievedthat theycausecell lysisvia

permeationofthecytoplasmicmembrane(ChungandKhanum2017).

However,thehightoxicityofAMPstomammaliancellsandtheirpoorstabilityhave

ledtopoorresultsinclinicaltrials.Thishasguidedresearchintothemodificationof

AMPs, to produce Structurally Nanoengineered Antimicrobial Peptide Polymers

(SNAPPs),whicharemorestableanddisplaygreaterlevelsofantimicrobialactivity

(Lametal.2016).Whileasyetunprovenclinically,SNAPPshavebeenshowntobe

particularlyactiveagainstGram-negativebacteriaandshowpromisingsynergywith

conventionalantibiotics.

Antimicrobialpeptides fromprokaryotesareknownas “bacteriocins”and tend to

haveanarroweractivity spectrum.Bacteriocinshavebeenused for some time in

foodpreservatives–forexamplenisinisusedtotreatcheese,meatandvegetables

to improve shelf life (Yang et al. 2014).While effective as inhibitors ofmicrobial

colonisation, the instability and potency of bacteriocins has to date limited their

potentialastherapeutics.

27

FigureRemoved

1.1.3.4 Bacteriophage

Bacteriophage,or“phage”,arethesubsetofvirusesthaninfectbacteria.Theyare

believedtobethemostnumerousbiologicalentitiesontheplanet,outnumbering

bacteria10-to-1anddisplayinghugevariability(Oliveiraetal.2013).Phage-therapy,

whichinvolvesusingphagetocombatbacterialinfections,hasbeeninvestigatedas

apotentialantibioticsincetheearly1900sbutinterestinthefielddeclinedacross

much of the globe after the discovery of penicillin. However, phage therapy

continuedtobepracticed in theUSSRdueto their limitedaccess toconventional

antibiotics,andeventodaytheEliava Institute inGeorgiastill treatspatientswith

phagetherapy(Fernebro2011).

Phage therapymakesuseof the lytic cycleof livebacteriophage to lyse infecting

bacteria.Thelyticcycle,illustratedinFigure1.5,involvesspecificattachmentofthe

bacteriophagetothehostbacterialcellwall,injectionofviralDNAintothehostcell,

andrecruitmentofthehostproteinsynthesismachinerytoproducebacteriophage

progeny.Aftersufficientbacteriophageprogenysynthesis,hostcelllysisisinitiated

bythecombinedeffectsofholinandendolysinproteins,thuskillingthehostcelland

releasingthephageprogenyintotheenvironment(ReindelandFiore2017).

Figure 1.5 The bacteriophage lytic cycle. Phage specifically bind to their target bacterialspecies and inject viral DNA. The host cell protein synthesis machinery produces phageprogeny,thenholinstoformaporeinthecellmembraneandfinallyendolysinstohydrolysethepeptidoglycanlayer.ReproducedfromFeineretal.(2015).

28

Sincetheriseofantibioticresistanceandthedecline innewantibioticdiscoveries

however,interestinphagetherapyhasbeenrenewed.In2006phagetreatmentof

cookedmeatswasapprovedbytheFDAtopreventpost-processingcontamination

with Listeriamonocytogenes (Fernebro 2011).However, no FDA approval has yet

beenmadeforhumantherapies,althougharecent“emergencyinvestigationalnew

drugapplication”was approved for the treatmentof apatient in SanDiego,USA

suffering from a multi-drug resistance bacterial infection, which resulted in the

successfultreatmentofthatpatient(LaFeeandBuschman2017).

Thehighspecificityofbacteriophageresultsinanarrow-spectrumofactivity.Thisis

excellentnews for theprotectionof commensal flora in thegut forexample,but

relieson rapidandeffectivediagnosis techniqueswhicharenot readily available.

Furthermore,occurrenceofresistancetophageishigh,andpoordataexistonthe

efficacy of treatment and their safety profile (Fernebro 2011). However, if these

obstacles can be overcome, then the sheer number, diversity and availability of

bacteriophagemakethemaveryattractivesourceofnovelantibiotics.

1.1.3.5 Bacteriophageendolysins

Manyofthehurdlesfacedbybacteriophagetherapycanbeovercomebytheuseof

their endolysins. These are the lytic enzymes which degrade the host bacterial

peptidoglycancellwallduringcelllysis.Theuseoftheseproteinsasnovelantibiotics

holds great promise and is the focus of this thesis. Endolysins are introduced in

greaterdetailinsection1.2.

1.2 Anoverviewofbacteriophageendolysins

1.2.1 Thebiologicalroleofbacteriophageendolysins

1.2.1.1 Background

Peptidoglycanhydrolase is a genericnamegiven to anenzyme that catalyses the

hydrolysisof thebacterialpeptidoglycancellwall.Peptidoglycanhydrolasescome

frommanydifferent sources,withdifferent functions:autolysinsareproducedby

bacteriatoremodeltheircellwallorcauseapoptosis;exolysinsareaweaponused

29

by bacteria to attack competing bacteria of another species; lysozymes can be

produced by eukaryotes as part of their innate immune response; tail-associated

lysins enable bacteriophage to inject their DNA into a new host bacterial cell;

endolysinsarepeptidoglycanhydrolases,alsoofviralorigin,thatlysethebacterial

hostcellwallfromwithinattheendofthelyticcycle.

Upon injectionofbacteriophageDNAintothehostbacterium,bacteriophageviral

progenyaresynthesizedandassembledbythehostmachinery.Twootherproteins

arealsoproduced,whichactinsynchronytooptimizethetimingofhostbacterium

lysis.Oneistheholin,whicholigomerisesintheinnermembranetoformapore.The

otherisanendolysinwhichisabletoaccessthehostpeptidoglycancellwall,viathe

poreformedbytheholins,degradingthecellwallandcausingcelllysis.

1.2.1.2 Structure

Endolysinsaregenerallyencodedbyasinglegeneandtwodistinctcategoriesexist:

those fromGram-positive infectingbacteriophage,and those fromGram-negative

infectingbacteriophage.EndolysinstargetingGram-positivebacteriahaveamodular

structure and arehighly species specific. Theirmodular structure consists of aN-

terminalcatalyticdomainandaC-terminalcellwallbindingdomain(CBD),generally

connectedbyaflexibleacidiclinker(Figure1.6).Themajorityofendolysinspecificity

is conferredby thecellwallbindingdomain,although in somecases thecatalytic

domain has been shown to bestow specificity via recognition of the enzymatic

substrate(Mayeretal.2011).RemovaloftheCBDinsomeinstancescanimprovethe

activityandspectrumofanendolysin,but inothercasestheCBDappearstobea

requirementforactivity,perhapsbyachievingthecorrectorientationofthecatalytic

domaintothesubstrate.

Figure1.6ThecanonicalmodularstructureofaGram-positiveendolysin.Thecellwallbindingdomain (CBD) is responsible for conferring specificity to the enzyme andmodification orremovaloftheCBDhassometimesresultedinimprovedactivityofthetruncatedendolysin(ChengandFischetti2007).

CellwallbindingdomainCatalyticDomain Acidic

linkerN C

30

Gram-negative endolysins on the other hand tend to be smaller with a globular

structure,andlackthehighbindingaffinityandspecificityforthecellwall.Thereason

for this is believed to be that, while it may be advantageous for Gram-positive

targetingendolysinstobindtightlytothepeptidoglycancellwallinordertoprevent

them from lysing surrounding bacteria whichmight be future hosts for the viral

progeny, the surrounding Gram-negative bacteria are protected by their outer

membranesonosuchrestraintisrequired(Loessneretal.2002).

Thesedescriptionsareofcoursesimplifications,andtherearemanyexceptionsthat

provetherule.ForexampleseveralstaphylococcalendolysinshavetwoN-terminal

catalytic domains, the streptococcal λSA2 endolysin has a central CBD with two

flankingcatalyticdomainsateachterminal,andPlyC,anotherstreptococcallysinand

oneofthemostactiveendolysinstestedtodate,isencodedbytwoseparategenes

formingamultimericlysin(Schmelcheretal.2012).

Afewthree-dimensionalcrystalstructuresofendolysinshavebeensolvedtodate,

butformationofcrystalshasproveddifficultpartiallyduetotheflexibleacidiclinker

betweenthedomains.However,T4lysozyme(SagermannandMatthews2002),T7

amidase(Chengetal.1994),PSAendolysin(Korndörferetal.2006),Cpl-1(Hermoso

etal.2003),CTP1L(Dunneetal.2016)andpartsofCD27L(Dunneetal.2014)have

allbeenelucidated.ThecrystalstructureofCpl-1willbediscussedingreaterdetail

in4.3.1.

1.2.1.3 Mechanismofaction

Endolysinsdisplayhugevariationintheirmechanismofaction.Whiletheyallattack

thepeptidoglycan,therehavebeenendolysinsidentifiedwhichtargetalmostevery

bondwithinthepeptidoglycannetwork.Thesecanbebroadlysplitintoglycosidases

whichhydrolysetheaminosugarlinkages,andamidases/peptidaseswhichattackthe

cross-linkingpeptidestemsandinterpeptidebridges(Loessner2005).

31

Figure1.7ThepeptidoglycanfromStreptococcalcellwall(A3α).Hexagonsrepresentsugarsandovalsareaminoacids.Redarrowsindicatesitesofactionforglycosidases(A:N-acetyl-β-D-muramidase;B: N-acetyl-β-D-glucosaminidase). Green arrows indicate endopeptidasetargets (C: D-glutamyl-L-lysine endopeptidase; D: D-alanoyl-L-alanine endopeptidase).ModifiedfromSchmelcheretal.(2012).

CBD domains are present in Gram-positive targeting endolysins, and recruit the

catalyticdomaintothepeptidoglycancellwall.TheCBDislargelyresponsibleforthe

specificityobservedinendolysinsandisabletorecognizeandbind(non-covalently)

arangeof ligands inthecellwall.These ligandscaneitherbeadirectpartof the

peptidoglycan,ortheycanbeothercomponentsofthecellwall.Anexampleofthe

firstistheLysMdomain,oneofthemostcommonconservedbindingmodules,which

binds the GlcNAc residues in the peptidoglycan. Meanwhile, the SH3b domain

presentintheCpl-1endolysinbindscholinemoietiesincell-wallassociatedteichoic

acidsofStreptococcuspneumoniae.Thespecificityconferredtoendolysinsbytheir

CBDcanrangefromanentirebacterialgenus,downtoserovarorstrainandoften

thebindingisexceptionallytight–inthepico-tonano-molarrange(Loessneretal.

2002;Briersetal.2009).

GlcNAc GlcNAcMurNAc MurNAc

D-Ala

GlcNAc MurNAc MurNAc

L-Ala L-Ala D-Ala

L-Lys

D-Glu

L-Ala

D-Glu

L-Lys

GlcNAc

NH2

H2N

L-Ala

A B

C D

32

FigureRemoved

1.2.2 Thetherapeuticpotentialofendolysinsand“exolysis”

1.2.2.1 Theoryandhistory

Gram-positive bacteria, which have no outer membrane, are also susceptible to

endolysins applied exogenously as the endolysin can directly access the

peptidoglycan cell wall. The osmotic pressure inside the bacterial cell (20-50

atmospheres) then causes cell lysis. This sensitivity enables endolysins to be

exploitedasnovelantibacterialagents,witharangeofadvantagesoverconventional

antibiotics.

Gram-negativebacteriaontheotherhandhaveaprotectiveoutermembrane(Figure

1.8) which prevents the endolysins from accessing the peptidoglycan layer when

appliedexogenously.However,recentadvancementshaveopenedupthefieldfor

endolysin therapy against Gram-negative bacterial infections. The addition of a

polycationic peptide, able to cross the lipopolysaccharide outer-membrane, to a

globular Gram-negative endolysin has yielded exciting results, and the chimeric

proteinshavebeennamedArtilysins®(Briersetal.2014).However,Gram-positive

targetingendolysinsarethefocusofthisthesis.

Figure 1.8 The cellwall structural differences betweenGram-positive andGram-negativebacteria. The noticeable differences are the thicker peptidoglycan cell wall in the Gram-postivebacteria,andthepresenceofanoutermembraneinGram-negativebacteria.Inbothinstances the cytoplasm is at thebottomof thediagram.Reproduced from (Brownetal.2015).

33

The first in vivo demonstration of endolysin activity was performed by Vincent

Fischetti’s group at Rockefeller University in 2001. The group used an endolysin

prophylacticallytopreventaStreptococcalinfectioninthemousepharynx,showing

thatafterasingletreatmentnostreptococcalbacteriaremainedintheoralpharynx

whileleavingtheindigenousoralfloraunaffected(Nelsonetal.2001).Sincethenthe

fieldhasgrownsignificantly,withonecommerciallyavailableendolysintherapeutic

andseveralmoreinthepipelineaswillbediscussedinthefollowingsection.

1.2.2.2 Currenttherapeuticuses

TheDutchcompanyMicreosproducedthefirstendolysin-basedtherapytoreceive

FDAapproval.Theproduct,namedStaphefektandmarketedunderthebrandname

GladSkin, has been approved for topical treatment of Staphylococcus aureus on

unbroken skin, treating diseases such as acne, rosacea, eczema and furunculosis

(Micreos).

AnothernotablecompanyinthetherapeuticendolysinfieldisContraFect.ContraFect

arealsodevelopinganendolysintherapyagainstStaphylococcusaureusnamedCF-

301,whichisinphaseIIclinicaltrialsandhasbeengrantedFastTrackDesignationby

theFDAhavingpreviouslybeenputonclinicalholdduetoconcernsovertrialdesign

(Parmley 2014).Notably, ContraFect are focusinguponendolysin purity,with the

intentionoftreatingbacteraemiasystemically.ContraFectalsohaveaGram-negative

endolysinatdiscoverystage(ContraFect).

Totheauthor’sknowledge,thesearetheonlytwoendolysin-basedtherapeuticsin

clinical trials. However, there are other companies such as Lysando, who have

licencedArtilysins®,withnewproductsinsight.

BothMicreosandContraFectuseE.coliastheirendolysinproductionplatform,and

ContraFect inparticularhavefocussedonengineeringtheirendolysintobehighly

expressed, soluble and readily purifiedwhen produced in E. coli (Parmley 2014).

While this appears to have been a suitable approach for producing endolysins

targetingGram-positivebacteria,itisunclearwhetherthesecompanieswillseekto

use an alternative expression platform for producing endolysins targeting Gram-

34

negativebacteria.SomeGram-negativetargetingendolysinshavebeenshowntobe

highly toxic toE. coli, suchasSPN9CC (Limetal. 2014), and thereforepotentially

renderE.colianinefficientproductionplatform.

1.2.2.3 Advantagesofendolysinsoverconventionalantibiotics

Endolysins hold great promise as novel antibiotics and they have numerous

advantagesoverconventionalantibiotics.

Firstly,endolysinskillbacteriaatasignificantlyfasterratethanthatofconventional

antibiotics.Indeed,purifiedlysinsinnanogramquantitieshavebeenshowntoreduce

107Streptococcuspyogenesbymorethan6logsinonlyamatterofseconds(Fischetti

2008).InvivomodelshaveshownthattheendolysinClySachievesa3logreduction

ofbacteriain30minutes,whiledaptomycintakes6hoursandvancomycin>24hours

to achieve similar levels of killing (Pastagia et al. 2013).Generally, endolysins act

much faster thanconventionalantibiotics,which forblood infections inparticular

suchassepsisandbacteraemia,couldbelifesaving.

The specificity of endolysins presents an enormous advantage over conventional

antibioticsiftheinfectingbacteriahasbeendiagnosed.Theadvantagesaretwo-fold:

firstly, a specificantibioticwillnotdisrupt thebody’s indigenous “good”bacteria,

whilekillingthepathogen.Thisminimisestheopportunityforsecondaryinfections

such as Clostridium difficile infection and yeast infections which are common

complications after a course of broad-spectrum antibiotics (U.S. Department of

HealthandHumanServices2013).Secondly,asthevastmajorityofbacteriainthe

bodyorenvironmentareunaffectedbytheendolysin,noselectivepressureisapplied

to them. This is in contrast to broad-spectrum antibiotics, which put a selective

pressureforresistanceuponallbacterialspeciesintheenvironment,thusincreasing

thechancesofresistanceemerging.Highspecificityisalsoalimitationofendolysins

however,aswillbediscussedinsection1.2.2.4.

Oneofthegreatestproblemsthatconventionalantibioticsfaceistheirlimitedability

tokillpersistercellsinbiofilms.Biofilmscanforminvariouslocations–fromthegut

lining, to water treatment plant equipment. Bacterial cells in biofilms are in a

35

dormant state, designed to withstand harsh environments and therefore do not

undergoreplication.Duetothemechanismofactionofconventionalantibiotics,such

asdisruptingcellwallformation,thesebiofilm-dwellingbacteriaareunaffectedby

them.Aftertreatmentwithantibiotics,theunaffectedbiofilmcellsarethenableto

recolonisetheenvironment.Endolysinsontheotherhandactivelylysebacterialcell

walls, regardless of the state of the cell and have been shown to be extremely

effectiveagainstbiofilms(Gutiérrezetal.2014).

Oneofthegreatestandmostoftenquotedadvantagesofendolysinsisthat,sofar,

bacterial resistance to them has been unreported, despite efforts to produce it

(Loeffler et al. 2001). It is believed that several factors contribute to endolysin-

resistancebeingarareevent.Firstly,thenaturalevolutionofendolysinshasledto

theadoptionofhighlyconserved targets in thepeptidoglycancellwall,whichare

essentialforbacterialviability(Fischetti2008).Secondly,theexogenousapplication

andactionofendolysinsavoidsmanyoftheresistancemechanismsobservedagainst

conventional antibiotics (efflux pumps and deactivating enzymes). Finally, with

regardstoreallifeapplications,thespecificityofendolysinsmeansthatnoselective

pressureisbeingappliedtonon-targetbacteria.

Itisinevitablethatoneday,ifendolysinsbecomewidelyused,thensomeresistance

will eventually be observed. In the case of conventional antibiotics, as we have

discussed, discoveringnewantibiotics requires serendipity andahugeamountof

time,andhasrecentlyprovenverydifficult.Incontrast,giventhediversityandsize

ofthebacteriophagepopulation,thereisahugestoreofendolysinstotapwhenthe

needarises.

Waterpollutionbyconventionalantibioticsisagrowingconcern.Whenantibiotics

arenotfullymetabolisedbythebody,thehighstabilityofsomeantibioticscanlead

them to remain active throughout the sewerage system and, eventually,

watercourses too. These low levels of antibiotics in the environment create ideal

conditions forresistanceemergence,and indeedantibiotic resistantbacteriahave

beendetectedinBritishwatercoursesforsometime(HughesandMeynell1974).The

proteinaceousendolysinsontheotherhandarerelativelyunstable,withahalf-life

36

ofabout20minutesinthebody,soareunlikelytosurviveintootherenvironments,

andgiventheirspecificityandlackofresistance,areaconsiderablylowerrisk.

Finally,thoughnotatrueadvantageoverconventionalantibiotics, istheabilityof

endolysins to act synergistically with antibiotics to reduce antibiotic usage and

improvetherapeuticefficacy.Forexample,theendolysinClyShasbeenshowntoact

synergisticallywithoxacillintokillMRSA invivo.Asoxacillinisacellwallsynthesis

inhibitor,andcausescelllysisbytheresultingupregulationofautolysins,itisbelieved

thatClySfurtherexacerbatesthedegradationofthecellwall,thuspromotingeven

morebacterial autolysin activity (Daniel et al. 2010). Such synergistic effectsmay

enable discontinued antibiotics to be reinstatedwhen usedwith endolysins, and

reduceoverallantibioticintakeorcourselength.

1.2.2.4 Limitations

Thebacteriaposing thegreatest threat tomankindhavebeengiven theacronym

ESKAPE by the Infections Diseases Society of America, standing for Enterococcus

faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni,

Pseudomonasaeruginosa,andEnterobacterspecies(Rice2008).Ofthese,thefinal

four are all Gram-negative. Conventional antibiotics currently in clinical trials are

skewedtowardsGram-positiveactivity.Onelimitationofendolysinsistheirinability

tocauseGram-negativelysisduetotheirinabilitytopenetratetheoutermembrane.

However,asmentionedearlier,Artilysins®havemadesomeprogresstowardsbeing

able to tackleGram-negative infections, and for topical applications EDTA can be

usedtopermeablisetheGram-negativeoutermembrane(BriersandLavigne2015).

Therearetwofactorstobeconsideredintermsofimmuneresponsetoapplication

ofendolysins.Thefirst istotheendolysin itself:endolysinsareaproteinandthus

maytriggeranimmuneresponseincludingantibodygeneration,potentiallylimiting

futureuseofthatendolysin.Anattempttoreducethepostulatedimmuneresponse

toCpl-1wasmadebyPEGylationoftheprotein,awell-knownmethodofminimising

immune response and increasing half-life, but this completely inhibited the lytic

activityoftheendolysin(Reschetal.2011b).However,ithasbeenobservedinvivo

thatalthoughantibodiesare indeedraisedagainsttheCpl-1endolysin inmice,on

37

reapplicationnoadverseeffectswereobservedandactivitywasonlyslightlyslowed

(Jadoetal.2003).Itispostulatedthattheendolysinactivityismuchfasterthanthe

host’s antibody mediated secondary immune response, and is thus largely

unaffected.

Thesecondimmuneresponseofconcernistothelargevolumeofbacterialcelldebris

rapidly released by endolysin activity. Two studies have been undertaken to

investigate this issue, with varying results: the first study found that the Cpl-1

endolysinproducedanaccentuatedcytokineresponsewhencomparedtothesame

treatmentusingvancomycin(Entenzaetal.2005);thelatterstudyobservednomajor

difference incytokineproductionbetweenCpl-1andamoxicillin(Witzenrathetal.

2009).Thisdiscrepancyisthoughttobeanissueofendolysindosing:whenahigh

dose is given, then the bacterial cellwall is fragmented thus producing a greater

response,comparedtoalowdosewhichmerelypuncturesthebacterialcellswith

minimalfragmentation(Fischetti2008).

Bioavailability of endolysins is a concern given their large size compared to

conventional antibiotics. In particular, intracellular bacterial infections such as

chlamydiaandtuberculosisareparticularlyhardtoaccess,andpresentachallenge

evenforconventionalantibiotics.However,arecentdemonstrationthatPlyCisable

to enter human throat cells and kill internalized S. pyogenes demonstrates the

diverseabilitiesoftheendolysins(Shenetal.2016).Inthisinstanceitappearsthata

specificcomponentofthethroatcellmembrane,aphosphatidylserine,grantsaccess

tothePlyCendolysin–howeverit isalsonotedinthestudythatotherendolysins

couldnotbeexpectedtofunctioninthisway.Thisfindingdoeshoweverraisethe

prospectthatevenintracellularbacteriamayeventuallybetreatedwithendolysins.

Theonlyendolysincurrentlyonthemarket,Staphefekt,isdesignedfortopicaluse

and isdeliveredeitherasacreamoragel (Micreos). It isunclearhowContraFect

intendtodeliver theirendolysindrug,howevertheirstudiesanddiscussionshave

mostlyfocussedaroundintranasalandaerosoldeliverymethods(Doehnetal.2013;

Loeffleretal.2003).Oraldeliveryofendolysinswillbeharder,astheproteinaceous

natureofendolysinsmeansthattheyaresusceptibletodigestionanddenaturingin

38

thestomach,andwouldneedtobeencapsulatedtoensurereleaseintotheintestine

forexample.

The final limitationofendolysins,whichwascontrastinglypresentedearlierasan

advantage, is theirspecificity.Thenarrow-spectrumoftargetbacteriakilledbyan

endolysinpresentsachallengeforcliniciansinthattheinfectionmustbeaccurately

diagnosedbeforeanendolysintreatmentcanbeprescribed.Thisdiagnosisisnotyet

easilypossible,butthepotentialtocreateendolysin“cocktails”,whichtargetallthe

mostcommonbacteriaassociatedwithacertaindisease,mayworkaroundthis.

To conclude, although various challenges havebeen laid in thepathof endolysin

research,nonehasyetpresentedaseriousflawtothetechnologyasatherapeutic,

andtechniquesareemergingtosolvethemajorityofissues.

1.2.3 Otherapplications

The remarkable ability of endolysins to identify and strongly bind highly specific

bacterialtargetshasproducedinterestinfieldsotherthandirecttherapeutics.

Forexample,workbytheLoessnergroupatETH,Zurichhasusedendolysinstocreate

rapid diagnostic tools for food safety. Their technology uses electrochemical

impedance spectroscopy (EIS) to rapidlydetectListeria cells binding toendolysin-

derivedCBDsimmobilizedonagoldscreenprintedelectrode.Thisenablesextremely

rapid, accurate, and sensitive detection of contaminating Listeria cells in food

products(Tolbaetal.2012).

Nelson’s group at theUniversity ofMaryland have designed two novel endolysin

basedplatforms,designedtoaidthebody’snaturalimmunesystem.Thefirstisthe

generation of Infection Site Targeted Antitoxin antibodies (ISTAbs), which are

endolysinCBDsfusedtospecificantitoxinneutralizingmonoclonalantibodies.The

CBDtargetstheantibodytothepathogencellwall,whereitsequestersthereleased

toxins, thus reducing the toxin load and furthermore, functionally opsonises the

bacterium for opsonophagocytic killing by the host immune system. The second

approach,namedInfectionSiteTargeteduniversalBridgingAntigen(ISTuBA),fuses

39

CBDs to antigens for which most people have existing immunity (e.g. childhood

vaccinetargets).Thisanchorstheantigentothepathogencellwall,anddirectsthe

existingimmunitytowardsthenewpathogen(Nelson2016).

Otherapplicationsincludeendolysinbaseddisinfectants,whichhavebeenshownto

be 1,000xmore active than virkon-S against Streptococcus equi over 30minutes

(Hoopesetal.2009).Foodbiopreservationandbiofilmeliminationhavealsobeen

showntobesuccessfulapplicationsofendolysins(Ajueboretal.2016).

1.3 Producingrecombinanttherapeuticproteins

Thesuccessfuladministrationoftherapeutic insulintoaseverelydiabetic14-year-

oldboy in1922markedthefirstuseofaproteinasapharmaceutical (Joshietal.

2007). Since then, the ability to produce proteins recombinantly in a number of

biological platforms has led to lower costs of production and the rise of protein

therapies.In1977Somatostatin,averysmallinhibitorpeptide,wasthefirsthuman

recombinantproteintobeproducedinEscherichiacoli,byGenentechInc(Brooker

2010). Other platforms have since been developed to meet the various protein

requirements, such as producing larger proteins, glycosylation patterns, and

disulphide bonds. Here we provide a very brief overview of some of the most

commonexpressionplatformsusedtoday.

1.3.1 Commonexpressionplatforms

E.coli,asalreadymentioned,wasthefirstrecombinantproteinexpressionplatform

andremainswidelyused.Duetomanyyearsofresearchanddevelopmentresulting

in an excellent understanding of E. coli genetics, genetic engineering of E. coli is

cheap,easyandextremelyfast.Highyieldsofrecombinantproteinscanbereadily

achievedandmanygenetictools,suchasinducibleexpressionsystemsandprotein

secretion,enableitsprecisemanipulation.However,thesystemisnotwellsuitedto

producinglargeproteins(over50kDa).Furthermore,theproteinscanforminactive

inclusion bodies, E. coli does not naturally glycosylate proteins, and endotoxin

contaminationrequiresexpensivedown-streampurificationprocedures(Guptaand

Shukla2015).

40

ChineseHamsterOvary(CHO)cellsareprobablythemostcommonlyusedexpression

platform for current therapeutic proteins, in particular for the production of

antibodies (Dumont et al. 2015). As amammalian system, CHO cells are able to

produce large proteins (over 100 kDa) and glycosylate and fold them correctly -

although they do produce some post-translational modifications that are not

expressed in humans, and subsequently against which humans have antibodies

(Dumontetal.2015).Onthewhole,CHOcellsareusedtoproducelargerandmore

complexbiologicsthanothersystems.Theirdrawbacks includepotentialDNAand

viralcontamination,veryhighcostsofproductionandloweryieldsthansomeother

systems(DemainandVaishnav2011).

Yeast,asasinglecellulareukaryote,sharessomeoftheadvantagesofamicrobial

platform, such as cheap growthmedia and high yields, as well as advantages of

mammalians cells, such as protein glycosylation and disulphide bond formation.

Yeast therefore presents a very attractive platform for recombinant protein

expression,althougha few issuesprevent it frombeingusing forallproteins.For

example,yeast’shigh-mannose typeN-glycosylationpatterncan lead toa shorter

protein half-life and reduced therapeutic efficacy, and its lack of necessary

chaperonescanoftenresultinpoorproteinfolding(Nielsen2013).

Plant-basedplatformshavelaggedbehindothersoverthepastdecades,despitethe

numerousadvantagestheyholdasarecombinantproteinproductionplatform.Plant

cellsareabletofoldcomplexproteinsandperformpost-translationalmodifications

including glycosylation. Either whole plants or plant cell cultures can be used as

expression platforms. Both are devoid of the endotoxins and potential viral

contamination of other platforms, but otherwise have different advantages and

pitfalls.Wholeplants,suchastobacco(Nicotianatabacum)arehighlyscalable,and

costeffectiveatscale.Transientgeneexpressioninleaftissueofwholeplantscanbe

achievedveryrapidlyandhasbeenusedtosuccessfullyproduceZMapp, theonly

successfuldrugproducedtotreatEbolaviral infectioninhumans,aswellasbeing

regularlyusedforproducinginfluenzavaccines(Yaoetal.2015).However,several

issues have limitedwhole plant use to date, including the relatively long time to

produce stable transgenic plants desired for long-term production, variable

41

expression levels,and issuesofbiocontainmentat largescale (ParkandWi2016).

Plantcellculturesoffersolutionstomanyoftheseissues,suchprecisemonitoring

andcontrolofgrowthconditions, consistent cell yieldsandbiocontainmentwhen

growninbioreactors.Severalplantcelllinesareusedroutinely,suchasthosederived

fromN. tabacum, aswell as those fromedible crops including rice (Oriza sativa),

carrot(Daucuscarota)andtomato(Lycopersiconesculentum)(XuandZhang2014),

and in 2012 taliglucerase alfa, the first plant-made pharmaceutical approved for

humanuse,wasmadeinacarrotcelllinetotreatGaucher’sdisease(Grabowskiet

al.2014).

Like plant cell cultures, microalgae offer many similar advantages over other

platforms and higher plants, but have additional advantages too.Microalgae are

photosynthetic (unlikecarrotcell cultures forexample),enabling thepotential for

using cheaper, less complex media that is less susceptible to contamination.

Microalgaealsogenerallygrowfasterthanplantcellculturesandaremorerobustin

termsofresistingstressesfrompHchangesandshearstress(SunandLinden1999).

Finally,thegenerationofnewtransplastomiclinesisfasterinmicroalgae(2-3weeks)

thanplantcellcultures(4-5weeks)(Govea-Alonsoetal.2017).

1.3.2 Microalgaeasexpressionplatforms

1.3.2.1 Whataremicroalgae?

The term microalgae can generally refer to any unicellular organism capable of

oxygenic photosynthesis, both eukaryotic (e.g. green algae) and prokaryotic (e.g.

cyanobacteria)(Walkeretal.2005).Microalgaearethereforeanextremelydiverse

groupoforganismsandcanbefoundinbothfreshandsaltwaterenvironments,as

wellasterrestrially.Figure1.9illustratesthelevelofdiversityobservedwithinthe

microalgae.

42

FigureRemoved

Figure1.9Aselectionofimagesdisplayingthediversityofmicroalgae.(A)Meridioncirculare(B) Merismopedia elegans (C) Acutodesmus acuminatus (D) Microcystis novacekii (E)Arthrospira jenneri (F) Dissodinium pseudolunula (G) Zygnema circumcarinatum (H)Actinocyclus normanii (I)Pediastrumduplex (J)Klebsormidiumdissectum (K)Diploneis (L)Chlamydomonas reinhardtii. A-F from http://nordicmicroalgae.org/. G-K fromhttp://www.algaterra.org/.Lfromhttp://protist.i.hosei.ac.jp/.Imagesnottoscale.

MicroalgaesuchasArthrospira(Spirulina)andAphanizomenonhavebeenusedasa

sourceoffoodforthousandsofyears(Spolaoreetal.2006),butonlyinthelast50

yearshavemicroalgaebeensubjecttocommercialcultivation.Thefirstcompanyto

produce Chlorella sorokiniana on a commercial scale was Taiwan Chlorella

ManufacturingLtd,whichwasfoundedin1964andstillproducesChlorellatoday,to

besoldasdietarysupplement(Spolaoreetal.2006).WhileSpirulinaandChlorella

remainpopularhealth-foodproducts,theabilityofmicroalgaetoactas“sunlight-

driven cell factories” (Chisti 2008) for producing high value products has spurred

researchinthefieldtowardsharnessingthefullpotentialofthesediverseorganisms.

43

1.3.2.2 Thehistoryofmicroalgaeasanexpressionplatform

Microalgae,andinparticularthegreenalgaChlamydomonasreinhardtii,havebeen

used as a model organism for many years for the investigation of the genetic

mechanismsbehindcellularprocesses(Harris2009).Microalgaegainedprominence

as a recombinant protein production platform with their potential to produce

biofuelswithout affecting food production - as a photosynthetic organism itwas

postulatedthatalgaecouldbegrowninareasunsuitableforcropproduction,thus

producing biomass from cheap substrates on unused land. However, despite the

successful production of biodiesel in microalgae, the economics of this strategy

currentlyfailtocompetewiththatoffossil-fuels(Scrantonetal.2015).

Despitethecurrentshortcomingsofmicroalgaefortheproductionofbiodiesel,the

platformstillholdspromiseforhigh-valueproteinsandhasseveraladvantagesover

higher-plants. Microalgae have the ability, like higher plants, to synthesize and

correctlyfoldhumanantibodies,andmanyareGenerallyRegardedAsSafe(GRAS).

Butinadditiontotheseattributes,microalgaecanbegrowninclosedcontainment

photobioreactors,removingtheriskofgeneflowintotheenvironmentviapollen;

genetically engineered strains can be produced in amatter ofweeks rather than

sometimesyearsforhigher-plants;andscaleupfrombenchtoproductionscaleis

straightforward (Franklin and Mayfield 2004). Microalgae therefore present an

extremelyattractiveplatformforrecombinantproteinexpression.

Severalspeciesofmicroalgaehavetodatebeengeneticallytransformed,including

C. reinhardtii,Haematococcus pluvialis, Chlorella and diatoms (Spicer and Purton

2016). H. pluvialis is a natural producer of astaxanthin, a carotenoid with high

economic and health-related value, and it has been successfully genetically

engineered to produce higher levels of astaxanthin (Steinbrenner and Sandmann

2006).ThegreenalgaChlorellavulgarisisasourceofseveralproductsbelievedtobe

beneficial as health supplements, including high-quality proteins, fatty acids, and

essentialaminoacids,andamethodofagrobacterium-mediatedtransformationhas

beenestablished(Chaetal.2012).

44

However,whilevariousspeciesofmicroalgaehavebeengeneticallymodified, the

specieswith themost advanced genetic toolkit isC. reinhardtii.C. reinhardtii has

been shown to express multiple recombinant proteins using a variety of

transformation methods, and in all three genomes (nuclear, mitochondrial and

chloroplast)(Scaifeetal.2015).ThepotentialofC.reinhardtiiwillbeexploredfurther

inthefollowingsection.

1.3.3 Chlamydomonasreinhardtiiasanexpressionplatform

1.3.3.1 AnintroductiontoChlamydomonasreinhardtii

Chlamydomonas reinhardtii is a unicellular green alga found widely in soil and

freshwaterandbelongstotheChlorophytadivisionofgreenalgae.Figure1.10shows

aschematicofaC.reinhardtiicellaswellasascanningelectronmicroscopyimage,

highlightingthemainmorphologicalfeaturesofthecell.Itisapproximately10μmin

diameter and has two flagella enabling phototaxis, and a large chloroplast filling

approximately 70% of the cell volume (Harris 2009). C. reinhardtii can grow

phototrophicallyor,inthepresenceofacetate,mixotrophicallyorheterotrophically.

Allthreegenomeshavebeensequenced(Vahrenholzetal.1993;Mauletal.2002;

Merchantetal.2010),andasimple(andcontrollable)sexualcycleexists inwhich

nucleargenesareinheritedaccordingtoMendelianrules,chloroplastgenesinherited

uniparentallyfromthemating-type(mt)plusparent,andmitochondrialgenesfrom

themtminusparent.Furthermore,thehaploidnatureofthenucleargenomeand

the fact thatmany nuclear genes are single copy (rather thanmembers of gene

families) makes the recovery and classical genetic analysis of mutants very

straightforward.ThishasledtoC.reinhardtiibeingreferedtoasthe“photosynthetic

yeast” (Rochaix1995).Given theseattributesand the fact that it is relatively fast

growing(5-8hourdoublingtime,dependingonnutrient,CO2andlightavailability

(Hosleretal.1989)), ithasbeenanattractivemodel for studyingphotosynthesis,

circadianrhythms,matingandotherprocessesformanyyears(Harris2009).

45

FigureRemoved

Figure1.10(A)TheChlamydomonasreinhardtiicellrepresentedasaschematicdiagramwithlabelledphysiological features (Shi2013). (B)AScanningElectronMicroscopy imageofC.reinhardtiiclearlyshowingthemainmorphologiesofthecellincludingthetwoflagella(Smithetal.1996).

1.3.3.2 GeneticengineeringofChlamydomonasreinhardtii

C.reinhardtiiisthemostgeneticallywellstudiedofthemicroalgae,andmethodsto

transform the nuclear, chloroplast and mitochondrial genomes have all been

46

developed.Thesexualcyclecanbepreciselycontrolled,enablinggeneticcrossesto

becarriedoutandintroducedtraitstobecombined(Grossmanetal.2003).

The nuclear genome can be transformed using a number of methods including

electroporationandparticlebombardment to randomly incorporateDNA into the

genome(JinkersonandJonikas2015).Antibioticmarkersandrescuingofnutritional

auxotrophshavebeensuccessfulmethodsfornucleartransformationselection,but

positionaleffectsleadtoaspectrumofgeneexpressionlevels(Rochaix1995).The

nucleargenomeisveryGCrich(64%),andcodonoptimizationhasbeenshownto

playan important role in geneexpression levels, improvingGFPexpression levels

approximately 5-fold (Fuhrmann et al. 1999). However, gene silencing and low

protein yields have been problematic for C. reinhardtii nuclear transformants,

althoughrecentwork,suchasthatbyBarahimipouretal.(2016)demonstratinghigh

yields of HIV antigen P24, show that these issues are being gradually overcome.

Furthermore, Lauersen et al. (2015) have created the pOptimized vector system,

which allows the rapid swapping of reporter genes, selectable markers and

regulatory elements to speed up the optimisation of nuclear protein expression,

whileScrantonetal.(2016)havedemonstratedthesuccessfulidentificationanduse

ofsyntheticnuclearpromoterstoimproveheterologousnucleargeneexpression.

The chloroplast on the other hand offers a more attractive target for genetic

modification. Very simple transformation processes exist, for example transient

pores can be formed by vortexing cell wall-lessmutants with glass beads, which

enablesDNAentrytothechloroplast(Kindleetal.1991;Maliga2014).Homologous

recombination then allows precise targeting of expression cassettes into the

plastome, and generally results in greater protein accumulation than nuclear

transformations(Franklinetal.2002).Furthermore,bytransformingphotosynthesis-

deficient mutants, antibiotic-free selection is possible by using the rescue of

photosynthesis as a selection mechanism (Wannathong et al. 2016). The precise

method of transformation is described in more detail in 2.3.13. Gene silencing,

thoughttobeadefencemechanismagainstviralDNAinsertionintothegenome,has

notbeenobservedintheC.reinhardtiichloroplast.However,thechloroplastdoes

notglycosylateproteins.

47

FigureRemoved

AsummaryofthestrengthsandlimitationsoftheC.reinhardtiichloroplastcompared

tothenucleuscanbeseeninFigure1.11.

Figure 1.11 The nucleus and chloroplast genomes represent the major locations fortransgene insertion and recombinant protein synthesis in C. reinhardtii. While themitochondrial genome has been transformed it is not commonly used for recombinantproteinproduction.ReproducedfromRasala&Mayfield(2015).

1.3.3.3 ThesuitabilityoftheC.reinhardtiichloroplastforendolysinsynthesis

Overonebillionyearsagoanendosymbioticevent,resultingintheencapsulationof

aphotosyntheticprokaryoteinsideanonphotosyntheticeukaryotichost,isbelieved

tohavebeenthegenesisofthechloroplastorganelle(Purton2007).Sincethenthe

prokaryoticgenomehasshedmuchofitsunnecessarygeneticmaterial,resultingin

a simplified circular plastome. The C. reinhardtii chloroplast, sequenced in 2002

(Mauletal.2002),contains99genesandhasalowGCcontent(34%),incontrastto

thenucleargenome.

Theprokaryoticbeginningsofthechloroplastresultinanenvironmentwellsuitedfor

the production of endolysins, which are naturally produced in bacteriophage-

infectedprokaryotes.Forexample,thechloroplastappearstocontainproteasesthat

48

aredirecthomologuesofprokaryoticproteases(Adametal.2006).Thisisexpected

toleadtoarespectablehalf-lifeoftheendolysinsinsidethechloroplast,astheyare

adapted to be relatively resistant to prokaryotic proteases. Furthermore, the

chloroplastisnon-glycosylating,whichmayoftenbeseenasadisadvantage,butfor

theproductionofendolysins,andotherprokaryoticproteins,isbeneficial(Almaraz-

Delgadoetal.2014).TheexpressionofendolysinstohighlevelsinE.colihasbeen

reported to have a detrimental effect upon bacterial cell growth, presumably

becauseoflow-levelactivityagainstitspeptidoglycancellwalldespitethespecificity

ofendolysinsandlackofholin.However,plantandgreenalgalchloroplastshavelost

thecellwallduringevolution,andextremelyhighlevelsofendolysinaccumulation

(~70%TSP)havebeenreported in thechloroplastofNicotiana (Oeyetal.2009).

Therefore, the prokaryotic environment, lack of glycosylation, and lack of

peptidoglycanstructuresmakesthechloroplastawellsuitedplatformforendolysin

production(Oeyetal.2009).TheC.reinhardtiichloroplasthassuccessfullyproduced

therapeuticproteins inthepast, includingendolysins,asummaryofwhichcanbe

seeninTable1.1.

C.reinhardtiihasbeendesignatedGRASstatusandthereisspeculationinthefield

thatthismayleadtoreduceddownstreamprocessingcosts,oreventheabilitytouse

crudeextractsofrecombinantproteintherapeutically(Almaraz-Delgadoetal.2014).

In fact, the twocompaniesclosest to therapeuticendolysinproductionhaveboth

been hampered by issues of product purity and solubility with their bacterial

production systems.ContraFect, attempting toproduceanendolysin for systemic

use,havesofarfailedtoreceiveFDAapproval,whileMicreoshaveinsteadoptedto

produce an endolysin for topical use, with less regulation around product purity

(Parmley2014).

To date, themajor limitation tomicroalgae as a recombinant protein production

platformhasbeentheirlowproductionyields.CHOcellsarecapableofproducing4-

6 g/L of recombinant protein (Wurm 2004), yeasts 9-12 g/L, E. coli 15-17 g/L,

transgenicanimals(e.g.goatmilk)upto23g/L,andfungalexpressionsystemshave

been reported to achieve 35 g/L (Demain and Vaishnav 2011). These are clearly

optimized yields, and differ depending on the protein in question. However, the

49

highest level of recombinant protein production inC. reinhardtii is 5% TSP under

laboratoryconditions(Manuelletal.2007),whichwouldequatetoapproximately8

mg/Lculturevolumewhenscaledtopilotscale (Gimpeletal.2015).As it is,pilot

scale production of recombinant bovine milk amyloid A (MAA) in C. reinhardtii

achieved 3.28 mg/L (Gimpel et al. 2015). It is clear from these data that the C.

reinhardtii platformhas a longway to gobefore being able to competewith the

platformsnamedabove.However,rateofdevelopmentcanbefast:between1986

and2004therecombinantproteinyieldofCHOcellsrosefrom50mg/Lto4.7g/L

(Wurm2004).Thisvast improvementwasachievedthroughavarietyof improved

techniques such as better selection strategies for high-productivity clones and

identificationandsubsequenttargetingtotransgenestotranscriptional“hotspots”

(Kimetal.2012).Furthermore,differentplatformshavedifferentproteinrecovery

rates,forexample,duetoinclusionbodiesinE.coli,productionratesmaybehigh,

but recovery rates and renaturation rates can be extremely low, leading to the

possibility that another platform with a lower headline production rate is more

suitable(Dataretal.1993).

WhiletheGRASstatusofmanymicroalgaegiveshopethatsubsequentpurification

ofproteinproductswillbecheaperandlessnecessaryincomparisonto,forexample,

E.coli,thereareotherlimitationstotheiruseasaproteinproductionplatform,such

asthepresenceofchlorophyll.Chlorophyllisaproblematicpigmentpresentinalgal

extractsthatrequiresfurtherpurification,althoughthiscanbeperformedrelatively

cheaplybyuseofcolumnchromatographywithactivatedcarbon(Santillan-Jimenez

etal.2016).

50

RECOMBINANTProtein Function Use Reference

HSV8-lsc HumanantibodyagainstglycoproteinDfromHerpessimplexvirus Therapeutic (Mayfieldetal.2003)CTB-VP1 ProteinVP1fromfoot-and-mouthdiseasevirusfusedtocholeratoxinB(CTB) Vaccine (Sunetal.2003)

hTRAIL Tumournecrosisfactor-relatedapoptosis-inducingligand Therapeutic (Yangetal.2006)M-SAA Bovinemammary-associatedserumamyloid Therapeutic (Manuelletal.2007)

CSFV-E2 Swine fever virus structural protein E2 Vaccine (He et al. 2007)

hGAD65 Humanglutamicaciddecarboxylase Diagnostics (Wangetal.2008)

VP28 Whitespotsyndromevirusprotein28 Vaccine (Surzyckietal.2009)

CTB-D2 D2fibronectin-bindingdomainofStaphylococcusaureusfusedtoCTB Vaccine(oral) (Dreesenetal.2010)

VEGF Humanvascularendothelialgrowthfactor Therapeutic (Rasalaetal.2009)

Pfs25andPfs28 Plasmodiumfalciparumsurfaceproteins Vaccine(oral) (Gregoryetal.2012)

αCD22PE40 AntibodyagainstDC22surfaceproteinfromB-cellsfusedtotwodomainsofanexotoxinfromPseudomonasaeruginosa Immunotoxin (Tranetal.2013)

CtxB-Pfs25 Plasmodiumfalciparumsurfaceprotein25fusedtoCTB Vaccine(oral) (Gregoryetal.2013)

TPS4 Bifunctionalditerpenesynthase,fortherapeuticterpenoidsynthesis Bioprocessing (Zedleretal.2015)

VHH Camelidheavychain-onlyantibodies Therapeutic (Markeyetal.2015)

Arah1 Majorpeanutallergen Therapeutic (Gregoryetal.2016)

Cpl-1andPal Streptococcuspneumoniaetargetingendolysins Therapeutic (Stoffelsetal.2017)

CD27L(1-179) Clostridiumdifficiletargetingendolysin Therapeutic Thisstudy

Table1.1AselectionoftherapeuticrecombinantproteinssuccessfullyproducedintheC.reinhardtiichloroplast.Modifiedandupdatedfrom(Stoffels2015),originallyadaptedfrom(Spechtetal.2010)and(Almaraz-Delgadoetal.2014).

51

FigureRemoved

1.4 Targets/applications

1.4.1 Streptococcuspneumoniae

Although S. pneumoniae is a common commensal bacterium found in the

nasopharynx,and isusuallybenign,colonisationand infectioncancauseahostof

diseases, including pneumonia, meningitis, febrile bacteraemia and otitis media

(Bogaert et al. 2004). Pneumococcal infection accounts for the death of

approximately1.2millionpeopleeveryyear,mostofthemchildrenundertheageof

five(ObaroandAdegbola2002).

Figure 1.12 Scanning electron microscope image of an encapsulated strain (R6) of S.pneumoniae,initsstreptococcalformwith2%choline(left)anddiplococcalformwithoutcholine(right).Scalebarsare2μm.Imagereproducedfrom(Paiketal.1999).

S.pneumoniaeisamesophilic,non-motile,Gram-positivebacteriumandwasnamed

after its role in pneumonia. It usually takes a diplococcal form but can form

streptococciinliquidmedium.Dependinguponthestrain,apolysaccharidecapsule

sometimesencasesthecell,belowwhichisthepeptidoglycancellwall.Presenceof

this capsule can increase virility of the strain and interferes with leukocyte

phagocytosis (Hyamset al. 2010).However, it hasbeen shown thatencapsulated

52

FigureRemoved

strainsarenomoreorlesssusceptibletolysisbyendolysinsthanun-encapsulated

strains(Loeffleretal.2001).

The peptidoglycan cell wall is approximately six layers thick and teichoic acid is

attached to approximately every third N-acetylmuramic acid. Specifically to S.

pneumoniae, both the teichoic acid and lipoteichoic acid (attached to the cell

membrane via a lipid moiety) are decorated with phosphoryl-choline residues.

Choline isanessential requirementforpneumococcalactivity,beingthe ligandby

which it attaches to human cells via the platelet-activating-factor receptor and

choline-bindingprotein,CbpA(DworkinandFalkow2006;Gehreetal.2008).Choline

isalsotheligandbywhichtheS.pneumoniaeautolysin,LytA,andtheendolysin,Cpl-

1,bindtothecellwall.

Figure1.13Aschematicofagenericpeptidoglycancellwall.SomestrainsofS.pneumoniaealso have an outer capsule, and both teichoic and lipoteichoic acids are decorated withcholine.ReproducedfromMadigan&Martinko(2006).

While highly effective vaccines have been produced to protect humans from S.

pneumoniaeinfections,theirsuccesshasbeenlimitedduetopoorcoverage,andthe

threat of non-vaccine serotypes requires an active solution (Bogaert et al. 2004).

Studiesagreethatpreventionofnasopharyngealcolonizationisthemostsuccessful

methodof averting infection,however the riseof antibiotic resistant strainsofS.

53

pneumoniae,inparticularpenicillin-resistantS.pneumoniae(PRSP),chloramphenicol

resistance,macrolide resistance, andmultiresistant strainspresentamajor global

healththreat(Appelbaum1992;Adam2002;Bogaertetal.2004;Staneketal.2011).

Thepresenceofavirulence-factortarget(choline)inS.pneumoniae,andtheriseof

multiresistant strains makes it an excellent candidate for endolysin treatment.

Furthermore,fornon-systemicinfections,forexampletopicalapplicationsorinhalers

to reducenasopharyngealcolonization, regulatory requirementsare less stringent

thusbeinganattractivetargetforendolysinproof-of-concept.

1.4.2 Clostridiumdifficile

Clostridium difficile, which inhabits the gastrointestinal tract, was first isolated in

1935(HallandO’Toole1935)andhashaditswholegenomesequenced(Sebaihiaet

al. 2006). It is a Gram-positive anaerobe, has the ability to form spores, and has

shownitselftobeparticularlyadeptatresistingantibiotics.C.difficileinfections(CDI)

generallyoccurnosocomially,andareacommonproblemafterantibiotictreatment,

causingantibiotic-associateddiarrhoeaandpseudomembranouscolitis.

11%oftheC.difficilegenomehasbeenshowntoconsistofmobilegeneticelements,

including conjugative transposons and phage, conferring high mobility to the

genome.ThishighlymobilegenomehasenabledC.difficiletosurviveinthehighly

variablegastrointestinaltractenvironment,andhasbenefittedfromanichewhere

opportunitiestoexchangeDNAwithotherorganismsarefrequent.Itisbelievedthat

manyoftheantibioticresistancegenespresentinC.difficileareencodedbythese

mobilegeneticelements(Sebaihiaetal.2006).Resistanceandmultiresistancehas

been reported to: metronidazole; vancomycin; clindamycin; erythromycin;

tetracycline;moxifloxacin;rifampicin;andfusidicacid,viaanumberofmechanisms

includingribosomalmodification,antibioticeffluxanddruginactivation(Huangetal.

2009). Furthermore, theabilityofC.difficile to formsporeswhen it comesunder

environmentalstresscauseshugehealthcareissues.Theresultantsporesarelong-

livedandresistanttomanydisinfectantsandantibiotics,makingthemasourceof

infectioninmanyhospitalenvironments(Gerdingetal.2008).

54

Spore-formation, alongside huge potential for antibiotic resistance, frequently

resultsinaresidualpopulationofC.difficilesurvivinginthehumangutafteracourse

ofantibiotics.Thispopulationisthenabletorapidlycolonisethegutduetolackof

competition fromnatural gut flora,and the result isCDI. Further treatmentusing

antibiotics isthenrequiredtotreattherecurrent infectionwhichcanincreasethe

rateoffuturerecurrence.Faecaltransplanttherapy,amethodofrepopulatingthe

gutwiththenaturalmicrobiotatocompetewithC.difficileandpreventCDI,aswell

asprobioticshavebeenusedrelativelysuccessfullytocombatthisissue(Allenetal.

2014).EndolysintherapyasatreatmentforCDIisparticularlyattractiveasitwould

notdisturbthenaturalgutfloraagainstwhichC.difficileisusuallyunabletocompete,

whileconventionalantibioticsoftenexacerbatethesituation.

1.4.3 Propionibacteriumacnes

PropionibacteriumacnesisaGram-positivebacilliformandformsasignificantpartof

thehumancommensalmicrobiome.Itcanbefoundontheskin,particularlyinfatty

acidrichareassuchasthefaceandscalp,aswellasoninternalmembranessuchas

thelargeintestineandurinarytract(DessiniotiandKatsambas2017).P.acnesisbest

knownforitsroleinthecommonskinafflictionacnevulgaris,butisalsoassociated

with a wide range of inflammatory diseases and implant-associated infections

(Portilloetal.2013).Acnevulgarisisoneofthemostcommonglobalskindiseases,

affecting45millionpeopleintheUSAandaccountingforapproximately20%ofall

dermatologist appointments (Bhatia et al. 2004). Currently treatments include

topical and systemic antibiotics, as well as chemical treatments such as benzyl

peroxide. However, growing antibiotic resistance and the sometimes severe side

effectsofchemicaltherapiescallforanewsolution.

Duetotheprevalenceandunsightlynatureofacnevulgaris,P.acneshasbeenwidely

treated with antibiotics for many years, and cross-resistance to clindamycin and

erythromycinwas first reported in1979.Sincethen, resistancetooxytetracycline,

doxycycline, andminocycline have also been reportedworld-wide (Dessinioti and

Katsambas2017).While rarely life-threatening in the formofacnevulgaris,other

less-commonP.acnesinfectionsbenefitfromresistancecommonlyacquiredwhen

55

treatingacne,forexample,implant-associatedinfectionsareaseriousthreat(Portillo

etal.2013).ItisthereforehighlydesirabletoreducetheselectivepressureuponP.

acnesduringthetreatmentofacne.

Asatopicalinfectionwithahighprevalenceofantibioticresistanceandlargeafflicted

population,acnevulgaris,andmorepreciselyP.acnes,makesasuitabletargetfor

endolysintherapy.Furthermore,thetargetedkillingofP.acnesintheaffectedregion

ispreferabletoeliminationofthebacteriumacrossthebody,asP.acneshasbeen

demonstratedtobeanonspecificimmunestimulant,somaybebeneficialatsome

level,althoughpreciselyhowthismechanismworksisnotyetunderstood(Bhatiaet

al.2004).

1.5 Summary,aimsandobjectives

The importanceofantibiotics tomodernsocietycannotbeoverstated.Theyhave

wideranginguses,fromenablinginvasivesurgeries,tolargescalefoodproduction.

Forthepast70yearswehavebecomeincreasinglydependentuponthem,butthe

efficacyoftheolddrugsisdwindlingandnewdrugsarebecominghardertofind.

A number of potential alternative antibiotics exists, each with strengths and

weaknesses.Fromreviewing the literature, ithasbecomeevident thatour future

antibiotic use will require a different strategy to that which we have become

accustomed.Wewillnotbeabletorelyonahandfulofbroad-spectrumantibiotics,

butarelikelytorequireavarietyofantimicrobialproductstobestsuittheproblem

inhand.

Oneof thepossible toolsagainstbacteria thatwewillbeable to callupon is the

bacteriophageendolysins.Thequestionremainsofhowtoproducetheminacost-

effectiveandsafemanner.RecombinantexpressionintheC.reinhardtiichloroplast

provides a potential solution to this problem: featuring GRAS status; lack of

glycosylation; quick and precise genetic transformation; and ability to scale to

productionrapidly.However,lowrecombinantproteinyieldshavesofarheldback

thisplatform.Threepathogens,S.pneumoniae,C.difficile,andP.acneshavebeen

selectedassuitabletargetsforendolysintherapy.

56

Thisthesissetsouttoaddresstheissuesdiscussedabove,withtheaimsofadvancing

boththeuseoftheC.reinhardtiichloroplastasarecombinantproteinplatform,and

thepotentialforendolysintherapy,usingthefollowingobjectives:

1. To improve the level of recombinant endolysin accumulation in the C.

reinhardtiichloroplast.

2. To improve the activity of endolysins produced in the C. reinhardtii

chloroplastagainstS.pneumoniae.

3. Todemonstratethesynthesisandactivityoftwoendolysinsneverpreviously

expressedintheC.reinhardtiichloroplast.

57

Chapter2 MaterialsandMethods

2.1 Strains,media,cultureconditionsandcelldensityquantification

2.1.1 Chlamydomonasreinhardtii

2.1.1.1 Strains

StrainsofC.reinhardtiiinthisthesisarenamedinasimilarmannertopreviouswork

(Taunt2013;Stoffels2015),andtheconventionisillustratedinFigure2.1.

Figure2.1AnamingconventionmodifiedfromTaunt(2013)andinlinewithpreviousworkby Stoffels (2015). This convention is used throughout this thesis, and any minormodificationsarenotedinthefollowingsections.

STRAIN DESCRIPTION REFERENCE

TN72 A recipient strain based upon the cell wall-less strain cw15 (mt+). Partial deletion of psbH gene via aadA cassette insertion. PSII-deficient mutant. Spectinomycin resistant.

Ninlayarn (2012)

TN72_SR_Control TN72 transformed with pSRSapI expression vector, containing no GOI, psbH rescued.

Young & Purton (2014)

TN72_SR_cpl1_c-HA

Thecellularbackground

Anabbreviationoftheexpressionvectorused.pSRSapI (SR),pASapI (A),

pWUCA2(WUCA)

Theexpressedgene

Modificationsandmutationsmadetothe

expressedgene

Epitopetag

58

TN72_ SR_cpl1-HA TN72 transformed with pSRSapI containing HA-tagged cpl1, psbH rescued.

Taunt (2013)

TN72_ SR_cpl1_CPO1-HA TN72 transformed with pSRSapI containing HA-tagged cpl1 with a single codon change, psbH rescued.

This study

TN72_ SR_cpl1_CPO2-HA TN72 transformed with pSRSapI containing HA-tagged cpl1 with a single codon change, psbH rescued.

This study

TN72_ SR_cpl1_W202A-HA TN72 transformed with pSRSapI containing HA-tagged cpl1 with tryptophan-202 mutated to an alanine, psbH rescued.

This study


This study


This study

TN72_ SR_cpl1_W202A_W209A-HA

TN72 transformed with pSRSapI containing HA-tagged cpl1 with tryptophan-202 and -209 mutated to alanine residues, psbH rescued.

This study

TN72_ SR_cpl1_W223A_W230A-HA

TN72 transformed with pSRSapI containing HA-tagged cpl1 with tryptophan-223 and -230 mutated to alanine residues, psbH rescued.

This study

TN72_ SR_cpl1_W202A_W209A_-W223A_W230A-HA

TN72 transformed with pSRSapI containing HA-tagged cpl1 with tryptophan-202, -209, -223 and -230 mutated to alanine residues, psbH rescued.

This study

TN72_ SR_cpl1_dimer-HA TN72 transformed with pSRSapI containing HA-tagged cpl1 with mutations to putatively enable dimerization, psbH rescued.

This study

59

TN72_ A_cpl1_c-HA TN72 transformed with pASapI containing HA-tagged cpl1 encoded by a different DNA nucleotide sequence (cpl1_c), psbH rescued.

This study

TN72_SR_cpl1-HA_A_cpl1_c-HA

TN72 transformed with a plasmid containing both the HA-tagged cpl1 under the psaA exon 1 promoter, and the HA-tagged cpl1_c under the atpA promoter, psbH rescued

This study

TN72_ SR_cpl1-StrepII TN72 transformed with pSRSapI containing StrepII-tagged cpl1, psbH rescued

This study

TN72_SR_cpl1-StrepII_A_cpl1-HA

TN72 transformed with a plasmid containing both the StrepII-tagged cpl1 under the psaA exon 1 promoter, and the HA-tagged cpl1-c under the atpA promoter, psbH rescued

This study

TN72_SR_cd27l-HA TN72 transformed with pSRSapI containing HA-tagged cd27l, psbH rescued

This study

TN72_ SR_cd27l-StrepII TN72 transformed with pSRSapI containing StrepII-tagged cd27l, psbH rescued

This study

TN72_ WUCA2_Control TN72 transformed with pWUCA2 expression vector, containing no GOI, psbH rescued.


TN72_ WUCA2_cph1-HA TN72 transformed with pWUCA2 containing HA-tagged cph1, psbH rescued

This study

TN72_A_gp20-HA TN72 transformed with pASapI containing HA-tagged gp20, psbH rescued

Taunt (2013)

TN72_ WUCA2_gp20N-HA TN72 transformed with pWUCA2 containing N-terminal HA-tagged gp20, psbH rescued

This study

60

2.1.1.2 Media

2.1.1.2.1 Tris-acetatephosphate(TAP)medium

NH4Cl 0.4 g/L

MgSO4•7H2O 0.1 g/L CaCl2•2H2O 0.05 g/L

K2HPO4 0.108 g/L KH2PO4 0.056 g/L

Tris 2.42 g/L Trace element stock solution 1 ml/L

Glacial acetic acid - to pH 7.0 Bacto agar (Optional) 2 g/L

2.1.1.2.2 Highsaltmedium(HSM)

NH4Cl 0.4 g/L

MgSO4 • 7H2O 0.1 g/L CaCl2 • 2H2O 0.05 g/L

K2HPO4 0.717 g/L KH2PO4 0.363 g/L

Trace element stock solution 1 ml/L HCl - to pH 6.9

Bacto agar (optional) 2 g/L

61

2.1.1.2.3 Traceelementstocksolution(Kropatetal.2011)

Adetailed recipe canbe found inKropatetal. (2011). Inbrief seven1000x stock

solutions were prepared and then combined into a single solution. 1ml of each

individualstock(7mlofmixture)wasaddedtoeachlitreofmedium.Thistracerecipe

wasdevisedtosupportacelldensityof2x107cells/ml(atypicalstationaryphasecell

density),withCu,Fe,MnandZnionsin3-foldexcessandmolybdatein2-foldexcess.

STOCK SOLUTION CONCENTRATION IN STOCK

COMPOSITION

EDTA-Na2 25 mM Titrated to pH 8.0 with trace element grade KOH

(NH4)6Mo7O24 28.5 µM

Na2SeO3 0.1 mM

Zn×EDTA 2.5 mM 2.5 mM ZnSO4 in 2.75 mM EDTA

Mn×EDTA 6 mM 6 mM MnCl2 in 6 mM EDTA

Fe×EDTA 20 mM 20 mM FeCl3 and 22 mM Na2CO3 in 22 mM EDTA

Cu×EDTA 2 mM 2 mM CuCl2 in 2 mM EDTA

2.1.1.3 Cultureconditions

Chlamydomonas reinhardtii was cultured by streaking the cells under aseptic

conditionsusingasteriletoothpickontoplatescontainingTAPsupplementedwith

2%bactoagar.Theplateswerethenincubatedat20°Cunderalightintensityof50

μmol/m2/s.

LiquidstarterculturesofC.reinhardtiiwerestartedin50mlflasks,with25mlliquid

TAPmedium.C.reinhardtiicellswerescrappedfromanactivelygrowingcolonyfrom

aTAPagarplateandsmearedontotheinsideoftheflask,justabovethewater-line.

Thecellswerethenresuspendedbyswirlingtheflasktoavoidclumping.Theflasks

were incubatedat25°C,120 rpmagitation,24-hour illuminationat50μmol/m2/s

62

lightintensity,tomid-logphase.A100-folddilutionwasthenperformedforlarger

sub-cultures.

2.1.1.4 Celldensityquantification

Cellcountingwasperformedusingahaemocytometer.1mlsamplewasextracted

fromaflaskand10μltinctureofiodine(19.7mMiodinein95%(v/v)ethanol)was

addedtoimmobilisethecells.Thenumberofcellsontwogridswasthencountedby

eyeat400xmagnification,themeantaken,andmultipliedby10,000toprovidean

estimationofthenumberofcells/mlofculturevolume.

Alternatively,theopticaldensityoftheculturewasmeasuredusingaUnicamUV/Vis

Spectrometerat750nm,withapath-lengthof1cm.

2.1.1.5 Storage

InoculatedTAPplateswerestoredat20°C,underdimlightconditions(5μmol/m2/s)

forupto5weeksbeforebeingre-streakedonfreshplates.

CryopreservationofC.reinhardtiistrainswasbaseduponaprotocolfromCrutchfield

etal.(1999).C.reinhardtiistrainswerere-streakedonTAPagarplatesevery3days

foraweektoensureactivegrowthstates.Thecellswerethenresuspendedin10ml

TAP-Sorbitol (1%),with the sorbitol acting as an osmoprotector for the cell-wall

deficientstrains,toaconcentrationofapproximately107cells/ml.Thesuspension

wasgentlyshakenat120rpmtoensurecelldissociationfor30minutes,andthen

dilutedto6x106cells/ml.Thecellsuspensionwasthengentlymixedina1:1ratio

withTAPcontaining10%methanol,underlowlightconditions.2mlaliquotsofthe

finalcellmixturewerethencooledatanapproximaterateof-1°C/minto-80°Cfor

90minutes,beforebeingstoredpermanentlyinaDewarcontainingliquidnitrogen.

63

2.1.2 Escherichiacoli

2.1.2.1 Strains


DH5α fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17

Hanahan (1983)

DH5α_ProEX_cpl1_dimer-HA-StrepII

Carrying pProEX HTb containing HA-tagged cpl1 with mutations to induce dimerization

This study

DH5α_ProEX_gp20-HA Carrying pProEX HTb containing gp20 tagged on the C-terminus with an HA tag

This study

2.1.2.2 Media

2.1.2.2.1 Lysogenicbroth(LB)(Bertani1951)

Bacto-tryptone 10 g/L Yeast Extract 5 g/L

Sodium chloride 10 g/L Bacto agar (optional) 15 g/L

Where necessary, ampicillin was added to a final concentration of 100 μg/ml.

Ampicillinstockswerecreatedat100mg/mlinH2Oandstoredinaliquotsat-20°C.


E.coliwasculturedeitheronsolidLBagarplatesat37°C,orinliquidLBat37°Cwith

constantshaking.

64

2.1.2.4 Storage

25%(v/v)glycerolstockswerepreparedforlongtermstorageofE.colibymixing

500μlofstationaryphasebacterialculturewith500μl50%(v/v)sterileglycerol.The

aliquotswerethensnapfrozeninliquidnitrogenandstoredat–80�.

2.1.3 Streptococcuspneumoniae

2.1.3.1 Strains


D39 Serotype 2, Mouse-passaged clinical isolate

McDaniel et al. (1987)

AL2 D39 LytA ̅ . Defined autolysin-deficient lytA mutant of D39; Emr

Berry et al. (1989)

2.1.3.2 Media

2.1.3.2.1 TrypticaseSoyyeast(TSY)extractmedium

Trypticase soy broth 30 g/L Yeast Extract 3 g/L

2.1.3.2.2 Columbiabloodagarplates(SIGMA)

Agar 15 g/L Special nutrient substrate 23 g/L

Sodium chloride 5 g/L Starch 1 g/L


S.pneumoniaewereculturedinliquidcultureinTSYmediumat35�inananaerobic

CO2richenvironment,usingananaerobic jar fromOxoidandOxoidAGCCO2Gen

Compactgaspacks.S.pneumoniaeculturesweregrownfor16hinTrypticaseSoy

yeastextractmedium,sub-culturedthenextmorningandgrowntomid-logphase.

InoculatedColumbiabloodagarplatesweresealedandincubatedat35°Cfor18h.

65

2.1.3.4 Celldensityquantification

Opticaldensitywasmeasuredin96-wellplatesusingaFLUOstarOmegaplatereader

(BMGLabtech)at595nm.

Serialdilutionfollowedbyacolonyformingunitassaywerealsousedtoestimatecell

density.

2.1.3.5 Storage

Aliquotsofbacterialcultureweresnapfrozeninliquidnitrogenandstoredat–80°C.

2.1.4 Clostridiumdifficile

2.1.4.1 Strains


NCTC 11204 Isolated from human neonatal meconium

(Hafiz and Oakley 1976)

2.1.4.2 Media

2.1.4.2.1 RobertsonsCookedMeatMedium(SGL,Corby)

Heart muscle 454.0 g/L

Peptone 10.0 g/L

`Lab-Lemco’ powder 10.0 g/L

Sodium chloride 5.0 g/L

Glucose 2.0 g/L

pH 7.2 ± 0.2 @ 25°C

66

2.1.4.2.2 LiquidBrainHeartInfusion+complements(Mayeretal.2008)

Brain infusion solids 12.5 g/L Beef heart infusion solids 5.0 g/L

Proteose peptone 10.0 g/L Glucose 2.0 g/L

Sodium chloride 5.0 g/L Disodium phosphate 2.5 g/L

pH 7.4 ± 0.2 @ 25°C Complements:

Vitamin K 50 mg/L Hemin 5 mg/L

Resaurin 1 mg/L L-cysteine 0.5 g/L


The C. difficile NCTC 11204 spore stock was stored in Robertson’s cooked meat

medium(SGL,Corby).Thestockwasthengrownin25mlofliquidBrainHeartInfusion

(BHI)+complements(Mayeretal.2008).After16hofgrowthat37°C,200-500μlofC.difficileculturewasusedtoinoculatefreshbrothandgrowntoanOD595nmof1-1.4

(latelogphase).

2.1.5 Propionibacteriumacnes

2.1.5.1 Strains


ATCC 6919 Type strain. Strain Designations: NCTC 737 [VPI 0389]

From Laura Stoffels

2.1.5.2 Media

2.1.5.2.1 LiquidBrainHeartInfusion

See2.1.4.2.2,withoutcomplements.

67

2.1.5.2.2 Columbiabloodagarplates(SIGMA)

See2.1.3.2.2.


P.acneswasgrownat37°Cfor4-5daysinliquidheartbraininfusionoronblood

agarplatesinananaerobicCO2richenvironment,usingananaerobicjarfromOxoid

andOxoidAGCCO2GenCompactgaspacks.

2.1.5.4 Storage

Aliquotsofbacterialsuspensionweremixedinequalvolumeswith50%(v/v)sterile

glycerolandsnapfrozeninliquidnitrogenbeforebeingstoredat–80°C.

2.2 Plasmids

PLASMID DESCRIPTION REFERENCE

pSRSapI Based on the pUC8 backbone, containing the endogenous psaA promoter and 5’ UTR, with the rbcL terminator and 3’ UTR. Includes flanking regions around the expression cassette for homologous recombination with the chloroplast genome, and a functional psbH gene

Young and Purton (2014)

pSRSapI_Control pSRSapI expression vector, containing no gene of interest


pSRSapI _cpl1-HA pSRSapI containing HA-tagged cpl1 Taunt (2013)

pSRSapI _cpl1_CPO1-HA pSRSapI containing HA-tagged cpl1 with a single codon change

This study

pSRSapI _cpl1_CPO2-HA pSRSapI containing HA-tagged cpl1 with a single codon change

This study

pSRSapI _cpl1_W202A-HA pSRSapI containing HA-tagged cpl1 with tryptophan-202 mutated to an alanine

This study

68


This study


This study

pSRSapI _cpl1_W202A_W209A-HA

pSRSapI containing HA-tagged cpl1 with tryptophan-202 and -209 mutated to alanines

This study

pSRSapI _cpl1_W223A_W230A-HA

pSRSapI containing HA-tagged cpl1 with tryptophan-223 and -230 mutated to alanines

This study

pSRSapI _cpl1_W202A_W209A_-W223A_W230A-HA

pSRSapI containing HA-tagged cpl1 with tryptophan-202, -209, -223 and -230 mutated to alanines

This study

pSRSapI _cpl1_dimer-HA pSRSapI containing HA-tagged cpl1 with mutations to induce dimerization

This study

pASapI Based on the pUC8 backbone, containing the endogenous atpA promoter and 5’ UTR, with the rbcL terminator and 3’ UTR. Includes flanking regions around the expression cassette for homologous recombination with the chloroplast genome, and a functional psbH gene

(Wannathong et al. 2016)

pASapI_cpl1_c-HA pASapI containing HA-tagged cpl1 with a different DNA nucleotide sequence (cpl1_c

This study

pSRSapI _cpl1-HA_A_cpl1_c-HA

pSRSapI containing both the HA-tagged cpl1 under the psaA exon 1 promoter, and the HA-tagged cpl1-c under the atpA promoter

This study

pSRSapI _cpl1-StrepII pSRSapI containing StrepII-tagged cpl1

This study

pSRSapI _cpl1-StrepII_A_cpl1-HA

pSRSapI containing both the StrepII-tagged cpl1 under the psaA exon 1 promoter, and the HA-tagged cpl1-c under the atpA promoter

This study

69

pSRSapI _cd27l-HA pSRSapI containing HA-tagged cd27l This study

pSRSapI _cd27l-StrepII pSRSapI containing StrepII-tagged cd27l

This study

pWUCA2 Based upon pSRSapI, with the introduction of a sythetic trnWUCA gene


pWUCA2_Control pWUCA2 expression vector, containing no GOI


pWUCA2_cph1-HA pWUCA2 containing HA-tagged cph1 This study

pWUCAC2_gp20N-HA pWUCA2 containing N-terminal HA-tagged gp20

This study

pProEX HTb An expression vector confering Ampicilin resistance and under trc promoter

Gift from J. Santini, Birkbeck College

pProEX_cpl1_dimer-HA-StrepII pProEX HTb containing cpl1 with mutations to induce dimerization. Tagged on the C-terminus with an HA-tag, followed by a StrepII tag

This study

pProEX_gp20-HA pProEX HTb containing gp20 tagged on the C-terminus with an HA tag

This study

2.3 Molecularbiology

2.3.1 Buffers

2.3.1.1 Sodiumphosphate(Na-Pi)buffer

NaH2PO4 59.99 g/L

Adjust pH to 7.4 with NaOH

2.3.1.2 Tris-HCl

Tris 121.14 g/L Adjust pH to 8.0 (unless otherwise stated) with HCl

70

2.3.2 Genedesignandsynthesis

GenesequenceswereacquiredfromGenbank(www.ncbi.nlm.nih.gov/genbank)and

verifiedwithBlastn. The sequence for an epitope tagwas addedbefore the stop

codon,unlessotherwiseindicated.

EPITOPE TAG AMINO ACID SEQUENCE

NUCLEOTIDE SEQUENCE

HA tag YPYDVPDYA

5' TAC CCA TAC GAT GTT CCA GAT TAC GCT 3'

StrepII tag WSHPQFEK 5’ TGG TCT CAC CCA CAA TTC GAA AAA 3’

His tag HHHHHH 5’ CAT CAC CAT CAC CAT CAC 3’

ASapIandaSphIrestrictionsitewere includedatthe5’and3’endsofthegene,

respectively,forsubsequentcloningunlessotherwiseindicated.Thesequenceswere

thencodonoptimisedusingCodonUsageOptimizerBeta0.92(Kong2013)software

developedspecificallyforC.reinhardtiichloroplastgenesinthePurtonlabbyKhai

Kong.GeneswerethensynthesisedbyIntegratedDNATechnologies.

2.3.3 Polymerasechainreaction(PCR)

DNAamplification,whencarriedout invitro,wasperformedusingthePolymerase

ChainReaction(PCR).Thereactionwasgenerallycarriedoutin50μlvolumes,and

usingtheFermentasPhusionpolymerase.Mix:

5x HF Buffer 10 µl

Template DNA 50-100 ng Primers 10 pmol each

20mM mixed dNTPs 1 µl Phusion polymerase 0.5 µl

ddH2O 35.5 µl

71

PolymeraseChainReactioncyclingparameterswerevariedslightlydependingupon

theprecisenatureofthereactionbeingperformed:theannealingtemperaturewas

calculateddependinguponthemeltingtemperature(Tm)oftheprimersbeingused,

and the extension time was altered depending upon the length of the expected

amplicon, according to the Fermentas Phusion polymerase instructions. As an

example,foramplifyingtheentireCpl-1geneusingaTechneTCF3000Xthermocycler

thefollowingconditionswereused:

98°C 2 min

98°C 10 s

X 30 cycles 60°C 30 s

72°C 30 s

72°C 5 min

10°C Hold

2.3.4 PCRDNAPurification

Following PCR, if the product was not to be loaded onto an agarose gel, it was

“cleanedup”bytheQiagenPCRclean-upkit,asperthemanufacturer’sinstruction.

Theclean-upremovedunwantedprimers,bufferandpolymerasefromthereaction

mixture. It was also used following restriction digests to remove small (<50 bp)

fragmentsofDNA.

2.3.5 AgarosegelElectrophoresis

1%agarosegelswereused for gel electrophoresis. 10μgofDNA sampleand1x

loadingdyewererunat10voltspercentimetreuntiltheladderiswellseparated.

EitherThermoScientificGeneruler1kbladderorGenerulerMixladderwereusedto

confirmDNAlength,forexpectedlargeorsmallbandsrespectively.

2.3.6 DNAgelextraction

Followingagarosegelelectrophoresis,DNAbandsofinterestwereexcisedfromthe

gelusingasterilescalpelandplacedintoamicrocentrifugetubeofpredetermined

72

mass.TheexcisedsliceofagarosewastheweighedandextractedusingtheQIAquick

GelExtractionKitfromQiagen,followingthemanufacturer’sinstructions.

2.3.7 SitedirectedmutagenesisbyPCR

PrimersweresynthesisedbyEurofinsandusedinconjunctionwithPolymeraseChain

ReactiontochangearequiredDNAnucleotide.

Figure2.2describestheprocess.

Figure2.2Processforsitedirectedmutagenesis.TemplateDNAisinblackandprimersareinblue.The‘X’indicatesapointmutation.Primersa+bandc+dwereinitiallyuseinseparatereactions.Thenthetworeactionswerecombinedand10cyclesofannealingwasperformed.Finally,afurther25cyclesofextensionwithprimersaanddwereperformed.

a b

c d

a

d

TwoseparatePCRreactions(a+b,c+d)

Anneal

PCRextensionandamplification

73

2.3.8 DephosphorylationusingAntarcticPhosphatase

InordertopreventtheligationoflinearizedplasmidDNAafterrestrictiondigestion

withouttherequiredDNAinsertion,the5’phosphatewasremoved.For1pmolof

DNAends,5unitsofAntarcticphosphatasewereaddedin1XAntarcticphosphatase

reactionbuffer.Afterincubationat37°Cfor30minutes,thereactionwasstopped

byheatinactivationat80°Cfor2minutes.

2.3.9 Blunt-endDNAcloning

Blunt-endDNAcloningintothepositiveselectioncloningvectorpJET1.2/bluntwas

carried out using a Thermo Scientific CloneJET PCR Cloning Kit according to the

manufacturer’sinstructions.

2.3.10 DNARestrictionendonucleasedigestion

LinearizationofthepSRSapIexpressionvector,andexcisionoftheGeneofInterest

(GoI) frompJetwas carriedout as shown in the tablebelow, followedby1hour

incubationat37°C.

Tango Buffer 4 µl

LguI (SapI) 1.2 µl PaeI (Sph1) 1 µl

DNA 2 µl

ddH2O 31.8 µl

2.3.11 DNALigation

DNAinsertandvectorconcentrationswereadjustedsuchthatthereisa3:1molar

ratioandapproximately20ngofvector.Then2μlof10xT4DNAligasebuffer,1μl

ofT4ligase(NewEnglandBiolabs)andddH2Oupto20μltotalvolumewasadded.

Thismixturewasthenincubatedatroomtemperaturefor1hour.

2.3.12 CompetentE.colitransformation–Heatshock

Competent E. coli (DH5α) stocks, stored at -80°C, were thawed on ice. For

transformationusingligationmixes,thewhole10μlligationreactionmixwasadded

74

to 100 μl of E. coli stock in a 1.5 ml microcentrifuge tube. For intact plasmid

transformations,approximately10ngofDNAwassufficient.Anadditionaltubeof

competent E. coli was also included as a negative control. The tubes were then

incubatedonicefor25minutes,followedbyaheatshockfor70sina42°Cwater

bath,followedbyanadditional2minonice.0.5mlofliquidLBwasaddedtoeach

tubebeforeincubationat37°Cfor1hourwithshaking.100μlfromeachtubewas

thenspreadontoLBamp100orLBkan100plates(dependingonantibioticresistance

geneintheplasmid)andincubatedat37°Covernight.

2.3.13 C.reinhardtiitransformation–glassbead

TN72isaphotosynthesis-deficient,cellwall-deficient,C.reinhardtiirecipientstrain

thatcanbegrownheterotrophicallyonmediacontainingacarbonsourcee.g.TAP

medium.Itisalsophotosensitivesoculturesgrowbetterwhenkeptinthedark.TN72

colonieswereregularlyrestreakedonfreshTAPagarplatesoverthreeweeksuntil

growinghealthilybeforestartinga400mlliquidTAPculture.

ThevectorpSRSapI (YoungandPurton2014)containsanexpressioncassette into

which a geneof interest can be inserted between SapI and SphI restriction sites.

pSRSapIcanthenrescuephotosyntheticactivity inTN72aftertransformationas it

carries thephotosystem II genepsbH,which isnon-functional inTN72.Successful

transformantscanthenbeselectedforonminimalmediaunderbrightlight,bytheir

abilitytogrowphototrophically.

Theprotocol followed forglassbeadtransformationwasbaseduponKindleetal.

(1991).TheTN72cellswereharvestedbycentrifugationat3,800xgfor15minutes,

oncetheculturedensityreached1-2x106cells/ml.Thepelletwasthenresuspended

inliquidHSMtogiveafinalconcentrationof2x108cells/ml.300μlofresuspended

cellsweretransferredtoanautoclavedglasstesttubecontainingapproximately300

mgof0.4mmdiameterpre-washedglassbeads.10μgofDNAwereaddedpertube

to11tubes,andonewasusedasanegativecontrol-withnoDNAadded.Thetubes

werevortexedfor15s,andthen3.5mlofwarmsoftHSMagarwereadded,quickly

swirledandpouredontoHSMplates.After20minutestheplateswereinvertedand

75

sealedwithParafilm,makingtwosmallpiercingsinthefilmwithasterilepipettetip.

Theplateswerethenplacedunderabrightlightfor21days.Allcoloniespresentafter

thistimeweretwicerestreakedontofreshHSMplates.

2.3.14 E.colicolonyPCR

E. coli colonies were picked using a sterile pipette tip and mixed well in a tube

containingTaqPolymerasePCRmixcontainingrelevantprimers.PCRwascarriedout

asdescribedpreviously,butwithanextrapreliminary2minutesofcelllysisat98°C.

Confirmedtransformantswerepickedagainfromtheplateandculturedforfurther

work.

2.3.15 E.coliplasmidisolation–CrudeandKit

Forplasmidpreparationsoflessthan12μgaQiagenMiniprepkitwasusedasper

the manufacturer’s instruction, while for larger preparations (<200 μg) a Qiagen

Midiprepkitwasused.

2.3.16 E.colitestdigest

Tango Buffer 1 µl

LguI (SapI) 0.2 µl PaeI (SphI) 0.2 µl

DNA sample 2 µl ddH2O 6.6 µl

A digestion was performed on plasmid mini/midi preparations as above and

incubatedat37°C for1hour.Gelelectrophoresiswasusedtoensurethecorrect

insertionhadbeenexcisedbydigestion.

2.3.17 C.reinhardtiigenomicDNAisolation

Thismethodallowscheckingforhomoplasmicity,thatis,toensurethatall~80copies

ofthechloroplastplastomehavebeensuccessfullytransformed.C.reinhardtiicells

werepickedusingasterilepipettetipandresuspendedin20μlofH2O.Then20μlof

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100%ethanoland200μlof5%CHELEXwereaddedandmixed.Afterincubationfor

10minutesina98°Cheatblockandthencentrifugationat13,000gfor10minutes,

the supernatant was used in a PCR using the following primers (unless stated

otherwise):

Flank1: GTCATTGCGAAAATACTGGTGC

rbcL.Fn: CGGATGTAACTCAATCGGTAG

RY-psaR: CATGGATTTCTCCTTATAATAAC (ForpSRSapItransformants)

atpA.R: ACGTCCACAGGCGTCGT (ForpASapItransformants)

Successfulrecombination:primersFlank1andRY-psaRgavea1,135bpproduct,and

Flank1andatpa.Rgavea1,204bpproduct.Parentalsequence(e.g.TN72):primers

Flank1andrbcL.Fngavean850bpproduct.

2.3.18 DNASequencing

SourceBiosciences carriedoutDNA sequencinguponprovidingDNA samplesand

primersaccordingtotheirinstructions.

2.4 Proteinanalysis

2.4.1 Preparationoftotalproteinextracts

TheOD740ofC.reinhardtiiculturewasmeasured.Theculturewasthencentrifuged

(3,795g,20minutes)andthepelletresuspendedinavolumecorrespondingtothe

obtainedOD.Forexample,a10mlC.reinhardtiiculture,withanODof0.569,would

beresuspendedin0.569mlofbuffer(20mMNaPibuffer+Proteaseinhibitorunless

otherwisestated).Then10μlof4xproteinloadingdye(200mMTris-HClpH6.8,8%

(w/v)SDS,40%(v/v)glycerol,4%(v/v)β-mercaptoethanol,50mMEDTA,0.08%

(w/v) bromophenol blue) was added to 30 μl of the sample to a final 1x

concentration.Themixturewas thenboiled for3minat98°Candcentrifugedat11,000gfor2min.Theresultingsupernatantwasusedasthepreparedsample.

77

2.4.2 Sodiumdodecylsulphatepolyacrylamidegels(SDS-PAGE)�

TheBio-Radmini-PROTEANTetraSystemwasusedtocastSDS-PAGEgels.Thegels

consisted of a 15 % polyacrylamide resolving layer, topped with a 3.75 %

polyacrylamidestackinglayer,asdetailedbelow.

RESOLVING GEL (FOR 2 GELS)

Acrylamide/bisacrylamide (Sigma)�40% stock, acrylamide:bisacrylamide = 37:1

3.75 ml

Resolving buffer (8x stock)�0.25M Tris, 1.92M glycine, 1% SDS, pH8.3

1.25 ml

10 % SDS 0.1 ml ddH2O 4 ml

10 % ammonium persulphate 0.375 ml

Tetramethylethlenediamine 3.75 μl

STACKING GEL (FOR 2 GELS)

Acrylamide/bisacrylamide (Sigma)�40 % stock, acrylamide:bisacrylamide = 37:1

0.47 ml

Stacking buffer (4x stock) 0.5M Tris-HCl (pH 6.8)

1.25 ml

10 % SDS 0.05 ml

ddH2O 3 ml 10 % ammonium persulphate 0.25 ml

Tetramethylethlenediamine 3.75 μl

RESERVOIR BUFFER (10X STOCK)

Tris 0.25 M Glycine 1.92 M SDS 1 %

HCl To pH 8.3

78

Thepreparedsample(20μlfor10-wellgelor12μlfor15-wellgel)and5μlofproteinladder(ColorPrestainedProteinStandard,BroadRange(11-245kDa),(NewEngland

Biolabs))wereloadedontothegelandruninreservoirbufferfor120minutesat120

V.

2.4.3 Proteinpreparationfordotblot

TheC. reinhardtii culturewas resuspended as for the SDS-PAGEpreparation. The

freeze-thawmethodwasthenusedtomechanicallybreakthecells(3xfreezingin

liquidnitrogen,thawingat35°C).Thenthesampleswerecentrifuged(16,200g,2

min) and 2 μl of supernatant was pipetted directly onto the Hybond-ECLnitrocellulosemembranes(GEHealthcare).Themembranewasallowedtodryfor10

minandthenimmuno-detectionwasperformedasbelow.

2.4.4 CoomassieBrilliantBlueRstaining

TheSDSgelwas incubated inCoomassieBrilliantBlueR solution for1hour,with

gentle agitation, followed bywashing in destaining solution overnight. Gelswere

photographedusingtheLiCorOdyssey®CLximagingsystem.

CoomassieBlueSolution:

Coomassie Brilllant Blue R250 2.5 g/L

Acetic acid 100 ml/L Methanol 450 ml/L

ddH2O 450 ml/L

DestainingSolution:

Acetic acid 100 ml/L

Methanol 450 ml/L

ddH2O 450 ml/L

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2.4.5 Westernblotanalysis

Semi-dry electrophoresiswas used to transfer separatedproteins fromSDS-PAGE

ontoHybond-ECLnitrocellulosemembranes(GEHealthcare).Initiallythegel,3MM

WhatmanpaperandnitrocelluloseweresoakedinTowbinbuffer(25mMTris,192

mMglycineand20%(v/v)methanol)atroomtemperaturefor10minutes.Atransfer

stackwasbuilt, consisting (inorder fromtop tobottom)of4 sheetsofWhatman

paper,SDS-PAGEgel,nitrocellulosemembrane,4moresheetsofWhatmanpaper.

ThiswasthenplacedintoaBio-RadTrans-BlotSDsemi-dryelectrophoretictransfer

system,andallairbubbleswererolledoutandexcessbufferremoved.Transferwas

carriedoutaccordingtothemanufacturer’sinstructions,ataconstantvoltageof20

V(FisonsFEC570powerpack)for1hour.

2.4.6 Immuno-detection

2.4.6.1.1 Antibodies

ANTIBODY SOURCE DILUTION

Primary antibodies

Anti-HA (polyclonal, rabbit)

Sigma Aldrich, #H6908 1:2,000

Anti-rbcL (polyconal, rabbit)

Gift from J. Gray,

University of

Cambridge

1:20,000

Anti-StrepII (polyclonal, rabbit)

Abcam, #ab76949 1:2,000

Secondary antibodies

Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight 488

ThermoFisher Scientific, #35552

1:20,000

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2.4.6.1.2 Protocol

Aftertransfer,thenitrocellulosemembranewasblockedin3%(w/v)skimmedmilk

powderinTBS-T(20mMTrisbase,pHadjustedto7.4with5MHCl,137mMNaCl,

0.1%(v/v)Tween-20)for1houratroomtemperature,orovernightat4°C.Thiswas

followedbyprimaryantibodytreatmentin1.5%skimmedmilkpowderinTBS-Tfor

1houratroomtemperaturewhileshakingat60rpm.Themembranewasthenrinsed

twiceinTBS-T,followedbya10minwashinTBS-Tat60rpm.Thesecondaryantibody

treatment was then applied 1.5 % milk solution, but diluted according to the

manufacturer’s instructions.Detectionwasperformedusinga LiCorOdyssey®CLx

SystemandanalysedusingOdyssey®ImageStudioLiteVersion5.5.

2.4.7 Proteinquantificationassay

Inordertodeterminethetotalsolubleproteinconcentrationofsamples,aBradford

assay was performed in a 96 well plate. This was performed according to the

manufacturer’s instructions (SigmaAldrich).Bovineserumalbumin (BSA)dilutions

were used to generate a standard curve. Then various dilutions of the unknown

samplewerepreparedtofindonewithaconcentrationintherangeof0.1-1.4mg/ml.

250μlofBradfordreagentwasaddedtoeachwellcontainingtheBSAorunknownsampleandtheplatewas incubatedatroomtemperaturefor10minutes.Aplate

readerwas used tomeasure absorbance at 595 nm. The net absorbance vs. the

proteinconcentrationofeachstandardwereplotted,andthenbycomparingthenet

A595valuesofthesampleagainstthestandardcurveitispossibletodeterminethe

concentrationofproteinintheunknownsample.

2.5 C.reinhardtiiproteinpurification

2.5.1 Crudeextractpreparation

C. reinhardtii cell cultures were grown to an OD740nm of 2 – 3 and harvested by

centrifugationat5,000gfor20minutes.Thepelletedcellswerethenresuspended

in20mMNaPi-buffer(NaH2PO4,thepHwasadjustedto6.9withNaOH),orBinding

buffer(20mMNaH2PO4,280mMNaCl,6mMpotassiumchloride,pH7.4)ifbeing

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usedforlatercolumnpurification,to50xconcentration.RochecOmplete,EDTA-free

proteaseinhibitorwasalsoadded.Thecellswereeitherbrokenthroughfree-thawing

(3cyclesoffreezinginliquidnitrogenandthawingina37°Cwaterbath),orviaglass

bead abrasion (1 g of 0.4 mm diameter glass beads was added per 10 ml of

concentratedcells,followedbyvigorousshakingat4°Cfor10minutes).Subsequently

the broken cell suspension was centrifuged at 21,000 g for 10 minutes and the

supernatantcollected.Thesupernatantisreferredtoascrudeproteinextract.

To further remove cell debris and insoluble proteins from the supernatant,

ultracentrifugationat100,000gfor1hourwasperformed,producingasupernatant

referredtoasultracentrifugedcrudeproteinextract.

2.5.2 Ammoniumsulphateprecipitation

Ultracentrifuged crude protein extracts were stirred on ice and powdered

ammonium sulphate was added until the desired percentage of saturation was

reached.After30minutesofstirringthesolutionwascentrifugedfor30minutesat

3,000g.Thepelletwascollectedforfurtheranalysisandtheprocesswasrepeated

tothesupernatanttocollectallthedesiredproteinfractions.

2.5.3 Anti-StrepIIpurificationcolumn

Ultracentrifugedcrudeproteinextracts,inbindingbuffer,werepassedthrougha1ml

StrepTrapHPHiTrap™column(GEHealthcare)at1mlperminuteusingaperistaltic

pump and eluted using 2.5 mM desthiobiotin in binding buffer, as per the

manufacturer’sinstructions.

2.6 E.coliproteinpreparation

Three different methods of protein preparation were used for E. coli-produced

recombinantproteins:

2.6.1 IPTGinduction

ForE.colitransformedwiththepProEXHTbplasmid,transgeneexpressionmustbe

inducedusingIsopropylβ-D-1-thiogalactopyranoside(IPTG).Anovernightcultureof

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E.coliwasdiluted1:100andgrownin2mlLB+amp100for4hoursat37°C.After4

hours,1mlwassnapfrozeninliquidnitrogentoprovidethe“uninduced”control.1

mlofLB+amp100+1mMIPTGwasthenaddedtothecultureandincubatedat20°C

overnight, to provide an induced culture which was then analysed by western

blottingorusedforfurtherinvestigation.

2.6.2 BugBuster™(Novagen)

BugBusterProteinExtractionReagentwasusedasaccordingtothemanufacturer’s

instructions.Inbrief,E.colicellswereharvestedbycentrifugation(16,000gfor10

minutes)andresuspendedinBugBusterreagent(2.5mlofBugBusterforevery50ml

of bacterial culture). The suspension was incubated for 10 minutes at room

temperaturewithgentleshaking.Centrifugationat16,000gfor20minutesat4°C

removedtheinsolublefractionandthesupernatantwasusedforSDS-PAGEanalysis

orpurification.

2.6.3 HighPressureHomogenizer

E.coliwereharvestedbycentrifugation(16,000gfor10minutes)andresuspended

in1/5thculturevolumeofNaPibuffer.Apre-equilibratedhighpressurehomogenizer

(StanstedFluidPower)wasusedtolysethecellsat14psiandwashedwithbuffer

betweenruns.Theresultingcellextractwascentrifuged(16,000gfor30minutesat

4°C)toremovecelldebrisandtheresultingsupernatantwasfiltersterilised(0.22

μm)beforebeingusedforcolonyformingunitassaysorSDS-PAGEanalysis.

2.6.4 Sonication

Overnight cultures of E. coli were harvested by centrifugation (16,000 g for 10

minutes).Theresultingpelletwasresuspendedin1/5thculturevolumeof10mMTris-

HClbuffer(pH8.0)andplacedinapolystyrenemicrocentrifugetube.Sonicationwas

performedusingaQ700cup-hornSonicator(QSonica).Thebathwaspre-cooledto4

°C and sonication performed at 100% amplitude for a total of fourminutes (30

secondson,30secondsoff).Thelysedcellswerecentrifuged(16,000gfor30minutes

83

at4°C)andthepelletandsupernatantanalysedbySDS-PAGEorusedfor further

analysis.

2.6.5 Disulphidebondformationinvitro

Whiledisulphidebondformationinvitrowasunsuccessfulinthisstudy,thefollowing

method was used, adapted from Hyvonen (2002). Sonication was used to lyse

inducedE.colicellsresuspended in10mMTris-HClbuffer (pH8.0)asdescribed in

2.6.4,andtheresultingsupernatantwasusedforthisanalysis.Tocreateanoxidising

environment conducive to disulphide bond formation, glutathionewas used as a

redoxreagent,bothinitsoxidisedandreducedforms(GSSGandGSHrespectively).

Solutionsof10mMGSSGandGSHweremadefreshfrompowderedstocks(SIGMA).

25μlofE.colicellextractwascombinedwithGSSGandGSHinordertoachievea

rangeofratiosbetweenthetwo.Ingeneral,thefinalconcentrationofGSHwas2-5

mM,with10-foldlessoftheoxidisedform.Themixtureswereincubatedovernight

at20°CbeforebeingseparatedbySDS-PAGEandanalysedbywesternblotting.No

β-mercaptoethanolwasincludedintheloadingdyeandthemixturewasnotboiled

beforerunningonthegel.

2.7 Antibacterialactivityassays

2.7.1 Colonyformingunitassay

2.7.1.1 Streptococcuspneumoniae

S.pneumoniaeculturesweregrownasdescribedabove.100μlofculturewasmixed

with endolysin samples and incubated at 37 °C for 1 hour. The mixture was

subsequentlydilutedinten10-folddilutionsteps.20μlofdilutionnumbers6-10were

spotted, in triplicate, onto Columbia blood agar plates and incubated at 37 °C

overnight.Thenumberofcoloniesineachspotwerecountedbyeyeandthenumber

ofcolonyformingunitspermlwascalculated.

2.7.1.2 Propionibacteriumacnes

CulturesofP.acnesweregrownanaerobicallyasdescribedabove.Thecultureswere

centrifugedat5,000gfor20minutesandresuspendedin20mMNaPibuffer.50μl

84

of bacterial suspension and 50 μl of filter sterilized cell lysate or buffer were

incubatedat37°Cfor0,30,60,90,120or150minutes.10μlsamplesweretaken

after the incubation time from each combination and spotted in triplicate onto

Columbia blood agar plates. After anaerobic incubation at 37 °C for one week a

comparisonwasmadesubjectivelybetweengrowthasindividualcolonygrowthwas

notvisible.

2.7.2 Turbidityreductionassay

2.7.2.1 Streptococcuspneumoniae

S.pneumoniaecultureswereharvestedbycentrifugation(15min,5,500g)andthe

cellswereresuspendedin20mMNaPi-buffertoanOD600nmof~0.8.

The turbidity reductionassayswereperformed in96-wellmicrotitreplateswitha

finalassayvolumeof300μl.AnELx808microplatereader(BIO-TEKINSTRUMENTS

INC.)measuredtheOD595nmeveryminutefor200minutes,including2sofshaking

beforeeachmeasurement,at37°C.

Unless otherwise stated, 20μl of antimicrobial sample,with varying total soluble

proteinquantitieswereaddedto280μlofsuspensionofthetargetbacterium.Asa

positive lysis control for S. pneumoniae turbidity reduction assays, 20 μl of

deoxycholate was added. As a negative control, samples were prepared from

TN72_SR_Control.

2.7.2.2 Escherichiacoli

E.coliculturesweregrowninLBtostationaryphaseandpelletedbycentrifugation

(3,800g,5min). The followingprotocol todisrupt E. colimembranes isbasedon

Nakimbugweetal.(2006):

ChloroformcontainingTris-HClbufferwaspreparedbyadding30%(v/v)chloroform

toTri-HCl(50mM,pH7.0)andshakingfor30min,200rpm,at25°C.Thetopphase

ofthismixturewasthenusedtoresuspendtheE.colipelletinitsoriginalvolume.

Thiswasthenshakenfor45minat25°Candthencentrifugedagain(8000g,20min,

4°C).ThepelletwasthenwashedwithTris-HClandresuspendedinTris-HClbufferto

85

anOD600nmbetween0.7-0.8.100Uofeggwhite lysozymewasusedperwell asa

positivelysiscontrol.

TheassaywasthenperformedasdescribedforS.pneumoniaeabove.

2.7.2.3 Clostridiumdifficile

C.difficilecellswereharvestedbycentrifugation(2minat5,000g)andresuspended

inavolumeof20mMNaPibufferequivalenttotheirOD(e.g.1.2mlifanOD595nmof

1.2wasmeasured).10-50μgofC.reinhardtiiextractin30μlfinalvolumewasadded

to270μlofpreparedC.difficilecellsinbuffer.Positivecontrolswere100U/wellof

mutanolysin and 30 μl of CD27LNiNTA-purified protein (unknown concentration)

fromtheMayerlab.ALabsystemsBioscreenC(ThermoFisherScientific)measured

theODevery2minutesfor18h,at37°C,inaThermoFisher100-wellhoneycomb

plate.

86

Chapter3 ImprovingaccumulationlevelsofCpl-1inthechloroplast

ofChlamydomonasreinhardtii

3.1 Introduction

3.1.1 IntroductiontobacteriophagelysinCpl-1

ThebacteriophageCp1was first isolated in1981, from throat samplesofhealthy

children(Rondaetal.1981).Cp1infectsthebacteriumStreptococcuspneumoniae,

whichcausesarangeofgloballysignificantdiseases.S.pneumoniae isacausative

agentofpneumonia,meningitisandfebrilebacteraemia,causingatotalannualdeath

toll of 1.6 million people, mainly young children and the elderly in developing

countries(WHO2007).

In1987the39kDaproteinofCp1wascharacterisedasamuramidaseandnamed

Cpl-1 (Garcíaetal.1987).Thesamestudynoted thatcholinewas required in the

pneumococcalcellwallforCpl-1tobeactive,andin2003thecrystalstructureofCpl-

1wasdetermined,showingtwoactivecholinebindingsites(Hermosoetal.2003).

Cpl-1,displayedinFigure3.1,isa339-residuepolypeptidechainconsistingofanN-

terminalcatalyticdomainandaC-terminalcellwallbindingdomain(CBD),joinedby

a10residueacidlinker.

Cpl-1belongstotheglycosylhydrolase(GH)25family,basedupontheaminoacid

sequencesimilarityof itscatalyticdomain,and isbuiltofmoduleshomologousto

LytC,amajorS.pneumoniaeautolysin(Monterrosoetal.2008).Thecatalyticdomain

displaysacharacteristicpeptidoglycanbindingsiteandadeepholeofhighlynegative

electrostatic potential located at the C-terminal end of the catalytic barrel that

constitutestheactivesite.

TheCBDisknowntobindthecholinemoietyintheteichoicandlipoteichoicacidof

theS.pneumoniaecellwall(seeFigure1.13),withtwoidentifiedaccessiblecholine

bindingdomainsontheprotein.Theadditionoffreecholine,orthesubstitutionof

cholineforethanolamineintheS.pneumoniaecellwall,inhibitsCpl-1activity.The

CBDiscomposedoftwowell-definedregions,namedCIandCII,containingatotalof

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sixhomologousrepeatsofabout20aminoacids.CIconsistsoffourrepeatsinaleft-

handed superhelical arrangement and includes both active choline binding sites,

whileCIIhastworepeatsarrangedasanantiparallelsix-strandedβ-sheet.

Figure3.1Cpl-1crystalstructure.N-terminalcatalyticdomain(green)islinkedviaaflexibleacidiclinker(orange)totheCIdomain(blue)andtheCIIdomain(pink).Theboundcholinemoleculesaredisplayedinsitu,inball-and-stickrepresentation.ImageproducedusingJmol(PDB1oba).

ProductionofCpl-1isattractiveduetoitsantimicrobialpotential.VincentFischetti

wasthefirsttoproposeusingCpl-1asanovelantibioticandhisgroupshowedits

efficacyinbacteremicmice(Jadoetal.2003).However,aspreviouslydiscussedin

1.1.2,theantibioticmarketisoneofnotoriouslylowmargins,andproducingbiologics

iscurrentlyanexpensiveundertaking.Thisisdueinnosmallparttotheexpensive

purificationprocess involvedinremovingantigensandendotoxinsassociatedwith

traditionalproteinproductionplatforms,suchasE.coli.GiventheGRASstatusofC.

88

reinhardtii,we speculate that purifying recombinant Cpl-1 from theC. reinhardtii

chloroplastmaynotberequiredtobeasrigorous,andthereforebecheaper.

3.1.2 Factorstoconsiderinproteinaccumulation

The relatively low levels of recombinant protein accumulation achieved in theC.

reinhardtiichloroplast,whencomparedtoexistingplatformssuchasE.coli,offeran

area for improvement. This chapter discusses two techniques investigated to

improveaccumulationoftheCpl-1proteinintheC.reinhardtiichloroplast.

Whenproteinaccumulationisconsidered,onemustbreaktheissuedownintotwo

key factors, protein synthesis and protein degradation, and investigate how the

balanceofthetwoisleadingtotheobservedaccumulation.

Figure3.2Proteinturnoveriscomposedofproteinsynthesis(transcriptionandtranslation)andproteindegradation.

TheliteraturehasdescribedthatanincreasedquantityofmRNA,i.e.byincreasing

levels of transcription, does not appear to improve the final levels of protein

accumulation(Coragliottietal.2011).GiventhepolyploidnatureoftheC.reinhardtii

chloroplast and the strength of promoters from chloroplast genes encoding

photosyntheticcomponents,onecanimaginethattherateofsynthesisoftransgene

transcriptsislikelytobehigh(Hosleretal.1989;Blowersetal.1990).Thesamestudy

pointstotranslation,andinparticularribosomebindingandtranslationelongation,

as likely limiting factors.WorkbyTaunt (2013) indicates thatCpl-1degradation is

minimalintheC.reinhardtiichloroplast.

Thischapter,therefore,investigatestwopotentialmolecularmethodsforimproving

proteinaccumulation.Firstly,constructscontainingtwosimilargenesunderdifferent

Transcription Translation Degradation

DNA mRNA Activeprotein Aminoacids

89

promoterswereinsertedintothechloroplastinordertopotentiallyovercomethe

process of Control by Epistasy of Synthesis (Choquet et al. 1998). Secondly,

continuing upon previous work by Taunt (2013), codon-pair optimization is

investigatedinordertoimprovetranslation.

ExperimentsandcalculationsbyTaunt(2013)estimatedthatamid-logcultureofC.

reinhardtiicanaccumulateCpl-1toapproximately8mgL-1,or17mgg-1dryweight.

A comparable endolysin, Pal, produced by Stoffels (2015) achieved slightly lower

levelsofaccumulation:5mgL-1or13mgg-1dryweight.Theseofferabenchmark

uponwhichweshouldaimtoimprove.However,inthisthesisdrycellweightwas

not considered, as generallywewere interested in relative improvements, rather

thanactualyieldsofprotein.However,movingforwardaclearunderstandingofdry

cell weights achieved and their reliable quantification will be important in

understandingthecommercialviabilityoftheplatform.

3.1.3 EpistasyofsynthesisintheC.reinhardtiichloroplast

IthasbeenreportedthatintheC.reinhardtiichloroplastthe5’UTRofpetA,which

encodestheapoproteinofcytochromef,mediatesitsautoregulationoftranslation

viainteractionwiththeC-terminaldomainoftheunassembledprotein(Choquetet

al.1998).ThisprocessiscalledControlbyEpistasyofSynthesis(CES)andeffectively

meansthatproteinshavetheabilitytocontroltherateoftranslationoftheirown

mRNA.Therefore,whenusinganendogenous5’UTRfortransgeneexpression,the

nativeassociatedproteinlevelsmaycontrolthelevelofthetransgeneproduct.

We hypothesize in this chapter that the CES process is limiting the level of

recombinant protein synthesis, due to the presence of associated endogenous

proteins.Byintroducingtwocopiesofcpl1andexpressingthemunderthecontrolof

different5’promoter/UTRsintheC.reinhardtiichloroplastweaimtoincreaseoverall

levelsofCpl-1accumulation.

90

3.1.4 CodonusageintheC.reinhardtiichloroplastgenome

Redundancyinthegeneticcoderesultsinmultiplecodonscodingforthesameamino

acid.Itiswellestablishedthatthereisadifferenceinpreferenceamongstorganisms

forwhichparticularcodoncodesforwhichaminoacid,andthisisknownascodon

bias.Byoptimizingnucleotidesequencessuchthatpreferredcodonsareused,itis

possibletoimprovegeneexpressioncomparedtoun-optimizedversions(Franklinet

al.2002).

Protein coding sequence analysis inEscherichia coli byGutman&Hatfield (1989)

revealedthatthereisalsoastrongbiasincodonpairs,inthattheydonotappearto

berandomlydistributed.This isbelievedtobeduetothevaryingcompatibilityof

adjacentaminoacyl-tRNAacceptorsattheAandP-sitesofaribosome.This,inturn,

ledtothesuggestionthatcodonpairscanbeoptimizedinasimilarwaytocodons,

andthusfurtherimprovegeneexpressionlevels.Indeed,Irwinetal.(1995)produced

evidencetosuggestthattheinfluencesofcodonpairchoicearemoreimportantthan

thechoiceofindividualcodonsinE.coli,exemplifiedbythefactthatACCandCUG

are frequently used codons, but ACC:CUG and CUG:ACC are translated at very

differentrates.

Previous work by Taunt (2013) recognised a similar, non-random distribution of

codonpairs intheC.reinhardtiichloroplastplastome.Onepair inparticularstood

outasappearingextremelynon-random,thephenylalanine:argininepair,UUU:CGU.

Under a random codon distribution, one would expect to observe this pair

approximately 28 times in the chloroplast plastome, but in fact it never occurs.

Interestingly, the UUU:CGU pair is present in the well expressed cpl1 gene. This

projectinvestigatestheimpactofthiscodonpairuponCpl-1expression.

3.2 Aimsandobjectives

1. Toidentifyandoptimizeamethodforcomparingproteinaccumulationlevels

betweendifferenttransgenicC.reinhardtiilines.

2. ToproducetransgeniclinesofC.reinhardtiiinwhichtwocopiesofcpl1are

introducedunderthecontrolofdifferent5’UTRsequences.

91

3. Toshowimprovedlevelsofproteinaccumulationinthenewtransgeniclines

comparedwithexistinglines.

4. Toproduce two transgenic linesofC. reinhardtiiwithdifferent codonpair

optimizationstrategies.

5. To compare protein accumulation levels of the two codon pair optimized

versions.

3.3 Resultsanddiscussion

3.3.1 Investigationintomeasuringproteinaccumulationlevels

The first hurdle to overcome when attempting to improve protein accumulation

levels in the C. reinhardtii chloroplast was to identify and optimize an accurate

methodforcomparingtheproteinaccumulationlevelsbetweentwocelllines.Given

thealmostimpossibletaskofstartingtwoormorecultureswithanidenticalnumber

ofcells,inthesamegrowthphase,andgrowingthemunderidenticalphysiological

conditions,itwasapparentthatasuitablenormalisationtechniquewasrequired.Of

particularconcernistheabilitytoestimatetheerrorintroducedateachstageofthe

proteinpreparationandquantificationprocess.

3.3.1.1 Quantifyingcellspermillilitreofculture

When comparing two ormore algal cultures, onemust be sure to inoculate the

culturewiththesamenumberofcells.Thisimmediatelyintroducesanopportunity

forerror,whichhereweaimtoquantify.Thereareessentiallytwopractisedmethods

to estimate the cell density of an algal culture – either direct counting using a

haemocytometerandmicroscope,orbyusingopticaldensity(OD)asaproxyforcell

number.Bothofthesemethodsareinvestigatedandthissectionexaminestheerror

associatedwitheachmethod.

Thefollowingsetofexperimentswerecarriedoutasfollows:threeflaskscontaining

400ml TAPmediumwere inoculatedwith equal starter volumes from a singleC.

reinhardtii starter culture. These were incubated and measured in the Algem

photobioreactorfor96hours.Afterthis,thethreeflasks(labelled:a,bandc)were

92

used to provide samples to test all of the subsequent techniques. Therefore, the

followingsetofexperimentswereperformedusingpreciselythesamealgalcultures,

enablingthemtobecompared.

Twoerrorswillbemeasuredwitheachtechnique.Firstly,theerrorbetweenidentical

samples–e.g.measuringthesamecuvettethreetimes.Thisgivesanindicationof

themachine/techniqueerror.Secondly,theerrorbetweenflasks,whichwillprovide

an understanding of the pipetting error, a combination of measuring out of the

mediumandinaddingthestarterculture.

93

3.3.1.1.1 AlgemPhotobioreactor

TheAlgemPhotobioreactorautomaticallymeasuresOpticalDensityat740nm,while

maintaining near identical physiological conditions in three separate flasks. By

startingthreeC.reinhardtiicultures,andcomparingtheirOD740over96hours,itis

possibletoseetheerrorintroduced.

Figure3.3ThreeflaskscontainingthesamevolumeofTAPmediumandthesamevolumeofstarterculture(400mlTAPmediumwith2mlofstarterculture)weregrownintheAlgemPhotobioreactorfor96hours.TheaverageOD740ofthethreeflaskswascalculatedandtheerrorbarsindicatethestandarddeviationbetweentheflasks.

InordertoestimatetheerrorintroducedbytheAlgemphotobioreactor,thestandard

deviation between every threemeasurements in flask Awas calculated. As each

measurementwastakenat30-minuteintervals,thenforthepurposesofestimation,

threeconsecutivemeasurementswillbeconsideredtobefromthesametimepoint.

Theaveragepercentageerrorcalculatedinthismannerwas1.99%.

0

0.5

1

1.5

2

2.5

3

0 20 40 60 80 100

OpticalDensity(740nm

)

Time(h)

94

Meanwhile, theaverageerrorbetweenthethreeseparatecultures,shownbythe

errorbarsinFigure3.3,was4.17%.

3.3.1.1.2 Opticaldensityerror

Threebiological replicatesofC.reinhardtiicultures,a,bandc,weresampledand

measuredthreetimeseachusingaUnicamUV/VisSpectrometer(ThermoElectron

Corporation,USA)at750nm(OD740):

Three different samples were then taken from the same flasks and their OD740

measuredinthesamemanner:

These results indicate that a Unicam UV/Vis Spectrometer (Thermo Electron

Corporation,USA)hasanaverageerrormarginof0.35%whenmeasuringtheexact

samesamplethreetimes.Takingdifferentsamplesfromthesameflask,atthesame

time-point,raisestheerrorto0.74%–potentiallyduetothedifferentsettlingofcells

and non-uniform distribution of cells through the culture. This is lower than the

Algemphotobioreactor,butherethetimepointsareidentical,whiletoaveragethe

Algemtheywere90minutesapart.It isexpectedthatthetrueerroroftheAlgem

photobioreactorissimilartothis,asitusesasimilartechnology.

Theaverageerrormeasuredbetweentheculturesis4.15%,virtuallyidenticaltothat

observedintheAlgemphotobioreactor.

Count Calculations1 2 3 St.Dev. %error

Culturea 252 322 267 36.86 4.38%b 223 281 220 34.39 4.75%c 248 275 220 27.50 3.70%

Av. 32.91 4.28%

OD750 Calculations1 2 3 St.dev. %error

Culturea 0.541 0.532 0.540 0.0040 0.25%b 0.479 0.499 0.480 0.0092 0.63%c 0.499 0.503 0.505 0.0025 0.17%

Av. 0.0052 0.35%


Culturea 0.532 0.569 0.538 0.0162 0.99%b 0.479 0.499 0.480 0.0092 0.63%c 0.499 0.503 0.505 0.0024 0.17%

Av. 0.0093 0.60%


Culturea 252 322 267 36.86 4.38%b 223 281 220 34.39 4.75%c 248 275 220 27.50 3.70%

Av. 32.91 4.28%


Culturea 0.541 0.532 0.540 0.0040 0.25%b 0.479 0.499 0.480 0.0092 0.63%c 0.499 0.503 0.505 0.0025 0.17%

Av. 0.0052 0.35%


Culturea 0.532 0.569 0.538 0.0162 0.99%b 0.509 0.499 0.522 0.0094 0.62%c 0.503 0.521 0.499 0.0095 0.63%

Av. 0.0117 0.74%

95

3.3.1.1.3 Haemocytometererror

AhaemocytometercanbeusedtocountthenumberofC.reinhardtiicellsinacertain

volumeandthenthatnumbercanbeusedtoestimatetheoverallconcentrationof

cellsintheculture,asexplainedin2.1.1.4.

Inorderto investigatetheerror introducedwhencountingC.reinhardtiicells,the

samesamplesasaboveweredilutedinaratioof1:4withsterileTAPmedia,andthen

iodinetincturewasaddedtoimmobilizethecells.

Tocalculatethetotalcells/mlofculture,thecountednumberwasmultipliedbythe

dilutionfactorandthenby10,000asaccordingtothemanufacturer’sinstructions–

thisdoesnotaffectthepercentageerror.

Theaveragestandarddeviationwithinthethreecountsofthesamesamplesis1.65

x106cells/ml,representing~4.3%error.

Meanwhile,theaverageerrorcalculatedbetweentheculturesis8.16%,almosttwice

thatoftheAlgemphotobioreactorandUnicamSpectrometer.

3.3.1.1.4 Celldensityquantificationconclusions

Theseinvestigationsindicatethatthehaemocytometerintroducesthelargestdegree

oferrortocelldensityestimations,andtheUnicamUV/VisSpectrometerintroduces

theleast.Thisismostlikelytobeduetotheslightlyobjectivenatureofcountingcells

onagrid,todecidewhetherthecellisonoroffthegrid,andalsothehumanerror

whencountinglargenumbers.Itcouldalsobeduetotheunevendispersalofcells

acrossthehaemocytometer,orasettlingofcellsintheflaskbeforethesamplewas


Culturea 252 322 267 36.86 4.38%b 223 281 220 34.39 4.75%c 248 275 220 27.50 3.70%

Av. 32.91 4.28%


Culturea 0.541 0.532 0.540 0.0040 0.25%b 0.479 0.499 0.480 0.0092 0.63%c 0.499 0.503 0.505 0.0025 0.17%

Av. 0.0052 0.35%


Culturea 0.532 0.569 0.538 0.0162 0.99%b 0.479 0.499 0.480 0.0092 0.63%c 0.499 0.503 0.505 0.0024 0.17%

Av. 0.0093 0.60%

96

taken.However,theAlgemphotobioreactorhasaleveloferrorataround2%,but

asmentionedearlier,this iscertainlyanoverestimationduetothefactthatthree

timepointsacross90minuteswereused,andwithaC.reinhardtiidoublingrateof

approximately5hours, this isasignificantperiodof time.Additionally, theAlgem

photobioreactorisabletoautomaticallymeasureuptofourculturessimultaneously

acrossanextendedperiodoftime.

Therefore,theUnicamUV/VisSpectrometerwillbeusedforcelldensityestimations

andcomparisonsfor infrequentandone-offmeasurements.Otherwise,theAlgem

photobioreactorwillbeusedwherepossible.

Themanualcountingmethodofthehaemocytometerstillhasavalueinofferinga

realnumberforcelldensity,butforthepurposesofcomparingtwoormorecultures,

anopticaldensityreadingissuperior.

3.3.1.2 Westernblotquantification

3.3.1.2.1 Anti-HAprimaryantibodyfordetectionandquantification

Toinvestigatetheerrorintroducedwhenquantifyingwesternblotsignals,thethree

separateculturesofTN72_SR_cpl1-HA,grownintheAlgemPhotobioreactor,were

preparedforwesternblotting.Equalvolumesfromeachculturewereloadedinthree

technical repeats onto an SDS gel and probed with anti-HA primary antibodies,

followedbyGoatanti-RabbitIgG(H+L)SecondaryAntibodyIRDye®800CW(referred

to henceforth as IRDye®). The resulting signal was quantified using the LiCor

Odyssey®CLxSystem.

97

Figure3.4ThewesternblotwasquantifiedusingOdyssey® ImageStudioLitev5.Thebarchart shows the raw data and percentage errors. The table shows the signal from eachreplicatewithineachcultureandthecorrespondingpercentageerror.

InFigure3.4averyhighpercentageerrorwasobservedbetweentechnicalreplicates,

despiteanequalvolumeofsamplebeingloadedontotheSDSgel.Itispossiblethat

differentquantitiesofproteinwereincludedineachreplicate,duetopipettingand

loadingerror.

3.3.1.2.2 Anti-RbcLprimaryantibodyasloadingcontrol

Inordertotesttheassertionthatpipettingandloadingerrorcausedthehighlevelof

error in Figure 3.4, the endogenous protein RbcL (which is the large subunit of

Ribulose-1,5-bisphosphatecarboxylase/oxygenase(RuBisCo),theproductoftherbcL

gene), was probed with an anti-RbcL primary antibody on the same blot and

CultureA CultureB CultureC

Replicate1 22800 59300 348002 50700 51900 274003 39400 35800 25500 Average

%Error 37.3% 24.5% 16.8% 26.2%


4632 Cpl-1

0

10000

20000

30000

40000

50000

60000

70000

80000


Signal(IRFluorescence)

(kDa)

98

quantifiedinthesamemanner.Ifloadingerrorisindeedtoblame,thenonewould

expecttoseecorrelatingvariationindetectionofthiscontrol.

Figure3.5ThewesternblotwasquantifiedusingOdyssey® ImageStudioLitev5.Thebarchartshowstherawdata.Thetableshowsthesignalfromeachreplicatewithineachcultureandthecorrespondingpercentageerror.

There appears to be little correlation between the RbcL andHA detection levels,

althoughsimilarlevelsoferrorareobservedbetweenreplicates.Thiscouldbedue

toanumberoffactors.Possiblytheblottingefficiencyvariedacrossthemembrane,

so proteins were unevenly blotted from the SDS gel onto the nitrocellulose

membrane.Itcouldalsobeduetounevenantibodydecorationoftheblot,suggesting

a longerblotting time isnecessary. Finally, thequantification techniqueusing the

CultureA CultureB CultureCHA RbcL HA RbcL HA RbcL

Rep.1 22800 20600 59300 22500 34800 156002 50700 19700 51900 24000 27400 49303 39400 23000 35800 18000 25500 17100 HAAv. RbcL Av.

%Error 37.3% 8.1% 24.5% 14.5% 16.8% 52.9% 26.2% 25.2%


4632

58 RbcL

Cpl-1

0

10000

20000

30000

40000

50000

60000

70000

HA RbcL HA RbcL HA RbcL


Signal(IRFluorescence)

(kDa)

99

ImageStudioLitesoftwarerequiresthedrawingofadiscreetboxaroundtheband,

whichrequiressomejudgementandintroduceserror.

3.3.1.2.3 Anti-PsbOantibodyasloadingcontrol

Tofurtherconfirmtheresultabove,thesameblotwasadditionalprobedwithaPsbO

primaryantibody,whichisexpectedtobindtoanotherphotosynthesisprotein:the

33 kDa subunit of the oxygen evolving system of photosystem II. As a highly

conservedprotein,theaimwastouseitsdetectionasaloadingcontrol.However,as

seeninFigure3.6,no33kDaproteincouldbedetected.

Figure3.6Furtherprobingwithananti-PsbOantibodyfailedtoprovideausefulbandfornormalization.

Failure todetectPsbO left thequestionofwhy suchhigh variationwasobserved

betweenwesternblot technical repeatsunanswered,buttheRbcLresultsuggests

thatusingasingleproteinasaloadingcontrolisunreliable.

3.3.1.2.4 TotalProteinStainasloadingcontrol

REVERT™TotalProteinStainisspecificallydesignedforwesternblotnormalization

and can be easily quantified using the LiCor Odyssey® CLx system, in a separate

channeltotheproteinofinterest.

Toinvestigatetheusabilityandaccuracyofthissystem,atestwasrunusingasample

ofTN72_SR_cpl1-HAinvariousdilutions,asshowninFigure3.7.

SampleA

46PsbO32

SampleB SampleC

(kDa)

100

Figure3.7REVERTTotalProteinStainanalysis.ThreedilutionsofTN72_SR_cpl1-HAextractwereseparatedbySDS-PAGEand transferred tonitrocellulosemembrane.Wholeproteinstained,analysed(A)washed,theprobedforHAtaggedproteins(B),thenwashedagainandre-probedusinganti-RbcL(C).ImagedwiththeLiCorOdyssey®CLxsystem.(D)Comparisonofthesignalfromwholeproteinstain,HAdetectionandRbcLdetection.Note:Wholeproteinstainisshownas1/20thtotalsoastoenablecomparisontootherbars.

Figure3.7showsthattheREVERT®TotalProteinStaindetectssimilarlevelsoftotal

proteinwithinthethreereplicatesofeachdilution,andappearstobewellcorrelated

with Cpl-1 detection at higher concentrations, but more variable at lower

concentrations. RbcL detection on the other hand yet again displays a poor

5846

32

2522

32

A

C

B46

D

5846

0

50000

100000

150000

200000

250000

300000

350000

100% 100% 100% 66% 66% 66% 33% 33% 33%

Relativeproteindetection

REVERTTotalProteinStain Anti-HA Anti-RbcL

anti-HA

anti-RbcL

REVERTTotalProteinStain

10x 6 x 3x

(kDa)

101

correlation to anti-HA tagged protein detection. This is most likely due to the

interaction of the RbcL protein and anti-RbcL antibody – for example, the RbcL

proteinmayrequiremoreboilingtofullydenatureandenabletheanti-RbcLantibody

tobind,andwearethereforeseeingerraticbinding.

Overall,thisanalysissuggeststhatREVERT®TotalProteinStainisagoodmethodof

normalisingwesternblotsignalsprovidedthesignalisrelativelystrong.Therefore,a

tentimes(10x)concentrationofC.reinhardtiicultures,atagrowthstagewithan

OD740ofapproximately0.750usingtheUnicamUV/VisSpectrometer,willbeused

forproteinquantificationpurposesunlessotherwisespecified.

3.3.1.2.5 Usingaproteinstandardforquantification

Aproteinstandardenablesobservedproteinaccumulationlevelstobequantifiedby

comparingthemtoastandardofknownconcentration.Furthermore,by including

the same standard in all western blot analyses, one can normalize between

membranes,ifforexampletheyhavebeenblottedforslightlydifferentamountsof

time. A standard encoding two ormore epitopes will also enable comparison of

differentlytaggedproteins.

PreviousattemptsatquantifyingaccumulationofHA-taggedrecombinantproteinin

C.reinhardtiiinthePurtonlabhaveusedaMultiTagprotein(AbCam)thatincludes

anHAepitope,oracommerciallyavailableHA-taggedprotein (CalciumRegulated

HeatStableProtein1(CARHSP1))asproteinstandardsagainstwhichtheHA-tagged

proteins inC. reinhardtii cell extracts have been compared. However, the results

obtained in this manner have been unreliable due to several factors including

degradation of the protein standards, the unknown efficiency of blotting to

nitrocellulosemembrane and unknown accessibility of the HA tag to the anti-HA

antibodies.Furthermore,estimated levelsofaproteinobtained inthisway,when

expressedas‘percentageoftotalsolubleproteinincellextracts’werenotsupported

byCoomassieBrilliantBluestainedSDS-PAGEanalysisoftheextracts(Stoffels2015)

andappearedtooverestimatethequantityofaccumulatedproteininC.reinhardtii

extracts.

102

3.3.1.2.6 ProducingabespokeCpl-1proteinstandard

Inordertominimisetheproblemsstatedabovewithcommerciallyavailableprotein

standards,abespokeCpl-1proteinstandardwasproducedinE.coli.

A slightlymodified version of Cpl-1was used (cpl1_dimer), for compatibilitywith

otherprojects,see4.3.2,andbothHAandStrepIIepitopetagswereincludedonthe

C-terminus.ThegenewascodonoptimizedforE.coliandsynthesizedbyIntegrated

DNATechnologies.NcoIandNotIrestrictionsiteswereusedtoclonethegene in-

frameintotheIPTGinducibleexpressionvectorpProEX-HTb.Theresultingplasmid

was sequenced to confirm the correct insertion and used to transform DH5α

competentE.colicells.

TheresultingE.colistrainwasnamedDH5α_ProEX_cpl1_dimer-HA-StrepII,and its

recombinant protein product, purified using StrepTactin affinity to the StrepII

epitopetagandquantifiedusingaBradfordassay,would,therefore,produceamore

reliableproteinstandardforquantifyingCpl-1accumulationinC.reinhardtiithanthe

Multi-tagorCARHSP1standards.

IPTGinductionwasperformed(2.6.1)andcpl1_dimer-HA-StrepIIaccumulationwas

confirmedbySDS-PAGEandCoomassieBrilliantBlueRstaining(Figure3.8).

Figure3.8WholecellextractswereseparatedbySDS-PAGEandtotalproteinstainingwasperformedusingaCoomassieBrilliantBlueRstain.Note:despitethenameCpl1_dimer,thisisastandard39kDaCpl-1monomer(thename“dimer”refersto itsabilitytodimerize innon-denaturingconditions,asdiscussedin4.3.2).

The IPTG-induced DH5α_ProEX_cpl1_dimer-HA-StrepII cells were lysed using

BugBuster™ (2.6.2). The StrepII epitope tag on the Cpl1_dimer-HA-StrepII protein

x10

32

46

x5 x2 x1 x0.25

x10

x5 x2 x1

IPTGInducedxculturevolume

Uninducedxculturevolume

Cpl-1_dimer(39kDa)

(kDa)

103

was then used for protein enrichment via a commercially available StrepTactin

column(ThermoFisherScientific).ThepurificationfractionsareshowninFigure3.9.

Figure 3.9 The various fractions involved using the StrepTactin purification columnwereseparatedbySDS-PAGEandstainedwithCoomassieBrilliantBlueRstain.

CoomassieBrilliantBluestaininginFigure3.9indicatesthatotherproteinsasidefrom

Cpl-1arestillpresentintheelutedfractions,sotheCpl1-enrichedelutionwasthen

further purified and concentrated by ammonium sulphate precipitation – the

fractionsofwhichareshowninFigure3.10.

Figure3.10CoomassieBrilliantBlueRstainedSDS-PAGE.The20–40%ammoniumsulphatefractionwaschosenasthefractionwiththemostCpl-1comparedtoothervisibleproteins.

CellLysate

32

46

Flow

through

Wash1

Wash2

Elution1

Elution2

Elution3

Cpl1(39kDa)

55

Cpl-1(39kDa)

40-60%

20-40%

0-20%

46

32

23

Ammoniumsulphatepercentagesaturation

(kDa)

(kDa)

104

Threedilutionsofthe20-40%ammoniumsulphatefractionwerethenquantified

usingaBradfordproteinassay.Absorbanceat595nm(A595)wasmeasuredusinga

Nanodrop2000,enablingverysmallvolumestobeused.Thesemeasurementswere

comparedtoastandardcurvemeasuredinthesameway,showninFigure3.11.The

calculations using the standard curve to measure the protein concentration are

showninFigure3.12.

Figure3.11ABradfordassaywasperformed,andthethreedilutionscomparedtotheproteinstandards.Eachcolouredpointshownonthisgraphistheaverageof4measurements.

y=0.0005x+0.0167R²=0.93536

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0 20 40 60 80 100 120

Absorbance(595n

m)

ProteinConcentration (ug/ml)

Standard 22%Dilution 27%Dilution 31%Dilution

105

Figure3.12BradfordAssaycalculations.TheequationwasderivedfromthelineofbestfitfromthegraphinFigure3.11.

ItwasinferredfromFigure3.12thatthestocksolutionofpurifiedcpl1_dimer-HA-

StrepIIhadaconcentrationof230±30μg/ml.

Thesynthesisofthisproteinstandardwillenablecomparisonofdifferentwestern

blot analyses aswell as compare theaccumulation levels of proteins taggedwith

differentepitopes,inthiscaseHAandStrepII.

3.3.2 Investigatingcontrolbyepistasyofsynthesisinproteinaccumulation

ThelowyieldsofrecombinantproteinaccumulationintheC.reinhardtiichloroplast

areamajorfactorimpedingitasaproductionplatform.Hereweattempttoimprove

proteinaccumulationlevelsofCpl-1byexpressingthecpl1geneunderthecontrolof

twodifferentpromoter/5’UTRs.

3.3.2.1 ExistingC.reinhardtiilines

ThissectionusestwoC.reinhardtiilinescreatedbyTaunt(2013)ascontrols.Oneis

anegativecontrolthatwillbeusedthroughoutthisthesis,namedTN72_SR_Control,

asshowninFigure3.13thisistherecipientstrainofC.reinhardtiitransformedwith

anemptypSRSapIplasmid.ThesecondC.reinhardtiilineisTN72_SR_cpl1-HAwhich

willbeusedasthe“benchmark”forthischapter-i.e.alinedisplayingamoderate

levelofCpl-1accumulationuponwhichweaimtoimprove.

A595AverageA595

St.Dev.A595

(y=0.0005x+0.0167) StockConc.Average

StockConc.St.Dev.

AverageProtein

StDev.Protein

Dilution1(22.3 %)

0.0500.0380.0430.039 0.0425 0.128 51.6 6.61 231.9 29.7

Dilution2(26.7 %)

0.0510.0530.0370.045 0.0465 0.155 59.6 9.21 223.2 34.5

Dilution3(31.2%)

0.0570.0530.0460.053 0.0523 0.088 71.1 6.22 228.3 20.0

Average: 227.8 28.1

106

3.3.2.1.1 C.reinhardtiilineTN72_SR_Control

Figure 3.13 TN72 transformedwith an empty pSRSapI plasmid. Unlike the original TN72recipient,thecontroliswildtypeforphotosynthesis,likethecpl1transformants,asthepsbHdeletionhasbeenrescued.

3.3.2.1.2 C.reinhardtiilineTN72_SR_cpl1-HA

Figure3.14TN72 transformedwith thepSRSapIexpressionplasmidcontainingHA-taggedcpl1.

3.3.2.1.3 Westernblotconfirmation

ToshowthatCpl-1accumulationoccursinTN72_SR_cpl1-HAandthatnoproteinof

a similar size is detected in TN72_SR_Control, whole cell extracts of each were

preparedandseparatedbySDS-PAGE.Ablotofthegelwasthenprobedwithananti-

HA primary antibody and an anti-RbcL primary antibody, followed by IRDye®

secondaryantibodies.TheLiCorOdyssey®CLxSystemwasthenusedfordetection.

The55kDaRbcLbandactsasanapproximateloadingcontrolinthisinstance,toshow

thatcellextractwaspresentinthenegativecontrollane.The39kDaCpl-1protein

canonlybeseenintheTN72_SR_cpl1-HAtransformedline.

trnE2 psbH

5’psaA 1 3’rbcL

TN72_SR_Controltransformant MluI

trnE2 psbH

5’psaA 1 3’rbcL

TN72_SR_cpl1-HAtransformant MluI

cpl1-HA

107

Figure3.15Westernblotanalysisofwholecellextracts.AColorPrestainedProteinStandard,BroadRange(NewEnglandBiolabs,Inc.)wasusedtoestimateproteinsize.

3.3.2.2 TheproductionofC.reinhardtiilineswithimprovedCpl-1accumulation

WehavehypothesisedthatCESlimitsthelevelofrecombinantproteinaccumulation,

duetotheuseofendogenous5’UTRsforrecombinantproteinexpressionintheC.

reinhardtiichloroplast.Byintroducingthegeneofinterestintothechloroplastinside

two different expression cassettes, we speculate that the level of protein

accumulationpercellcanbeimproved.Inthiscase,wewillusetheendogenouspsaA

exon15’UTRandtheatpA5’UTR,whichhavebothbeenshowntoachievemoderate

levelsofrecombinantproteinaccumulationintheC.reinhardtiichloroplast(Taunt

2013).

However,giventhatwewishtotransformC.reinhardtiiusingtheconvenientTN72

system for targeted insertion into the chloroplast, we have only one plastome

insertionsite;namely,theneutralsitebetweenpsbHandtrnE2.Thetwoexpression

cassettes,containingthesamegeneofinterest,will,therefore,needtobeadjacent

to one another in the transformation plasmid, and be integrated by a single

transformationeventintotheplastomebyhomologousrecombination.

Thegeneof interest in this instance iscpl1which is~1kb in length.Homologous

recombination between DNA direct repeats in the C. reinhardtii chloroplast is

efficient–indeed,itisthispropensitytorecombinewhichweexploitforthetargeted

insertionoftransgeneswhenperformingC.reinhardtiichloroplasttransformations.

TN72_SR_

Control

4632

58 RbcLCpl-1

TN72_SR_

cpl1-HA

(kDa)

108

However, two identical copies of cpl1 in close proximity to one another are

considered tobeat veryhigh riskofundergoing recombinationbetween the two

copies,resultinginaplastomerearrangementandlossofonecopyoftheGeneof

Interest(GOI)(Figure3.16).Muddetal.(2014)suggestthatdirectrepeatsof~600bp

ormoreresultinveryhighfrequenciesofhomologousrecombination.Additionally,

sucharecombinationeventwouldstillproducerecombinantprotein,somaygoun-

noticedifitoccursinatransformantlinesubsequenttomolecularanalysisconfirming

thetwo-GOIinsertion.

Figure3.16Twoexpressioncassettesinparallelorientationmighthomologouslyrecombineto leaveaviableexpressioncassette in the chloroplastplastome, thus still produceCpl-1protein.

However, as illustrated in Figure 3.17, by arranging the expression cassettes in

antiparalleldirections,recombinationeventswillmerelyresultinaswitchingofthe

promoterregionsandnolossofDNAwilloccur.

5’psaA 1 cpl1-HA 3’rbcL5’atpA 3’rbcLcpl1-HA

cpl1-HA 3’rbcL5’atpA

cpl1-HA

109

Figure3.17Whenarranged in antiparallel directions, the recombinationeventwouldnotresultinanylossofDNAfromthetransformedplastome.

Therefore,theantiparallelconfigurationwasusedwhentransformingC.reinhardtii

withtwoexpressioncassettesinasingleplasmid,inparticularwhenthetwogenes

are highly similar. Furthermore, Coragliotti et al. (2011) report no detectable

differenceinexpressionasaresultoftransgeneorientation.Infact,theC.reinhardtii

chloroplastcontainstwolargeinvertedrepeatregionswhichallowsimilar“flip-flop”

recombination of the entire plastome, and it is believed that the two possible

orientationsexistinapproximatelyequalproportion,withnoapparentadverseeffect

(Harris2009).

3.3.2.2.1 C.reinhardtiilineTN72_A_cpl1_c-HA

However,toreducethechanceofDNArecombinationeventsbetweenthedirectly

repeated cpl1 genes, one gene was differently codon optimized to reduce the

sequence similarity. Thedifferent versionofcpl1wasnamedcpl1_c,with the “c”

standingforcodonchanged.

3.3.2.2.1.1 Cpl-1_cgenedesign

The original genetic sequence for cpl1 as used by Taunt (2013), and present in

TN72_SR_cpl1-HA,wasuploadedtoCodonUsageOptimizerBeta0.92(Kong2013)

software and codonsweremanually edited to ones of similar “scores” for theC.

reinhardtii chloroplast. In total 148nucleotideswere changedoutof 1050 (14%)

withoutaffectingtheoverallscoreforcodonusage.SapIandSphIrestrictionsites

wereincludedforcloning.

5’psaA 1 cpl1-HA 3’rbcL5’atpA3’rbcL cpl1-HA

cpl1-HA 3’rbcL5’psaA 1 5’atpA3’rbcL cpl1-HA

110

3.3.2.2.1.2 ExpressioncassetteproductionandE.colitransformation

Thefull-lengthcpl1_cgenewassynthesizedby IntegratedDNATechnologies.SapI

andSphIrestrictionenzymeswereusedtoclonethegeneintothepASapIexpression

vectorin-frame,producingtheplasmidpASapI_cpl1_c-HA.DH5αcompetentE.coli

cells were transformed with the resulting plasmid and a plasmid midi-prep was

performedtoproducesuitablequantitiesofplasmidforglassbeadtransformation.

3.3.2.2.1.3 C.reinhardtiitransformation

ThepASapI_cpl1_c-HAplasmidwasusedtotransformrecipientC.reinhardtiistrain

TN72via theglassbeadmethodasdescribed in2.3.13.Figure3.18 illustrates the

resultingC.reinhardtiicellline,TN72_A_cpl1_c-HA

Figure3.18TN72recipientstrainsuccessfullytransformedtocarrycpl1_cundertheatpA5’UTRinthechloroplast,andwithpsbHfunctionalityrestored.

Theplastomesoftheresultingtransformantswereconfirmedtobehomoplasmicas

describedin2.3.17.Inthisinstance,thethreeprimersusedwereFlank1,atpa.Rand

psbH.R. Figure 3.19 shows the resulting PCR products separated by gel

electrophoresis.TN72wasusedasanegativecontrol.Asafinalstep,thesequence

integrity of the cassettewas confirmed by DNA sequencing of the PCR-amplified

plastomeregion,asin2.3.18.

trnE2 psbH

3’rbcL

TN72_A_cpl1_c-HAtransformant MluI

5’atpA cpl1_c-HA

111

Figure3.19PCRscreeningforsuccessfultransformationofTN72andreachingahomoplasmicstate.(A)Schematicshowingtheproductexpectedwitheachprimercombination.(B)Gelelectrophoresis of thePCRproducts confirms that the transformant is homoplasmic as itlacksthe350bpbandoftherecipientstrain.

3.3.2.2.1.4 Westernblotquantification

AwholecellextractofTN72_A_cpl1_c-HAwaspreparedandseparatedbySDS-PAGE.

Afterblottingtonitrocellulosemembraneasdescribedin2.4.5,themembranewas

112

probedwithanti-HAandanti-RbcLprimaryantibodies,followedbyIRDye®secondary

antibodiesandfluorescencewasobservedusingtheLiCorOdyssey®CLxsystem.

Figure3.20WesternblotanalysisshowsthatsimilarlevelsofCpl-1proteinaccumulationaredetectedinthetwoC.reinhardtiilines.RbcLisanapproximateloadingcontroltoshowthatsimilar quantities of cell lysate were loaded onto the gel and a negative control,TN72_SR_Controlisalsoincluded.

TheresultsshowsimilarlevelsofCpl-1proteinaccumulation,andtheRbcLloading

controlconfirmsthatsimilarquantitiesofcelllysatewereused.

TN72_SR_

cpl1-HA

TN72_A

_cpl1_c-HA

Cpl-1(39kDa)

TN72_SR_

Control

RbcL58

48

32

0

5000

10000

15000

20000

25000

TN72_A_cpl1_c-HA TN72_SR_Control TN72_SR_cpl1-HA

Signal(IRFluoresence)

Anti-HA Anti-rubisco

TN72_SR_cpl1-HA TN72_A_cpl1_c-HATN72_SR_Control

Anti-HA Anti-RbcL

113

3.3.2.2.2 C.reinhardtiilineTN72_SR_cpl1-HA_A_cpl1_c-HA

Toseeifhigherlevelsofproteinaccumulationcouldindeedbeachievedwithtwo

differentcpl-1expressioncassettesinsertedintotheC.reinhardtiichloroplast,the

SR_cpl1-HAandtheA_cpl1_c-HAcassetteswerecombinedintoasingleplasmidand

usedtotransformC.reinhardtiirecipientstrainTN72.

3.3.2.2.2.1 Expressionplasmidproduction

The finalassemblyofpSRSapI_cpl1-HA_A_cpl1_c-HAwasachievedasdescribed in

Figure3.21.TheexistingMluIsiteinpSRSapI_cpl1-HAwasusedtoopenthevector,

andtheinsertwascreatedbysite-directedmutagenesisPCRto introduceaBssHII

restrictionsiteat the3’endof theA_cpl1-c-HAcassette.AsMluIandBssHII form

compatible restriction sites, the cpl1-c containing expression cassettewas ligated

intotheopenvector,producingoneconservedMluIsite.Theresultingplasmidwas

called pSRSapI_cpl1-HA_A_cpl1_c-HA. It is believed that the alternative

arrangement,i.e.openingthepASapI_cpl1-c-HAplasmidandinsertingtheSR_cpl1-

HA cassette,would have also been possible, butwhen bothwere attempted the

cloningsucceededwiththeformerfirstandthereforethisroutewascontinued.

The primers used for the PCR reaction are indicated below. The BssHII site is

underlined,andthemutatednucleotideisshowninlowercase.

Cpl1.F:CAGGCAATTTGCTTACACCTTT

Cpl1_BssHII.R:TTAAGATAAgCGCGCTACATCC

114

Figure3.21Schematicshowingthestages in theconstructionofpSRSapI_cpl1-HA_cpl1_c-HA. (A)Site-directedmutagenesisPCRwascarriedoutuponpASapI_cpl1_c-HAtoamplifytheexpressioncassetteandtointroduceapointmutationtocreateaBssHIIrestrictionsite.Theredarrowsrepresenttheprimersusedforthisreaction.(B)ThePCRproductresultingfrom(A)wasdigestedwithMluIandBssHIIbeforebeingcleanedupusingaPCRpurificationkit. (C) pSRSapI_cpl1-HA was digested using MluI before being treated with AntarcticPhosphatasetopreventre-circularizationoftheplasmid.(D)Theresultinginsert(A_cpl1_c-HA)andvector(pSRSapI_cpl1-HA)werethenligatedusingT4DNAligase.

DH5αE.coliweretransformedwiththeresultingplasmid.Sequencingconfirmedthe

successfulinsertionandorientationoftheinsert.

MluI (7035)

pASapI_cpl1_c-HA

A

B C

D

pSRSapI_cpl1-HA

MluI (7035)

0 200 400600

8001000

1200

14001600

18002000

2200

2400

2600

2800

3000

3200

3400

3600

3800

4000

4200440046004800

50005200

5400

5600

5800

6000

6200

6400

66006800

7000

7200

7400

7600

7800

8000

8200

8400

8600

88009000

9200

MluI (2065)

pSRSapI_cpl1-HA_A_cpl1_c-HAMluI (7035)

MluI (5979)

3’RbcLcpl1_c-HA

5’atpA

5’psaA 1

cpl1-HA

3’RbcL

psbH

115


ThepSRSapI_cpl1-HA_A_cpl1_c-HAplasmidwasusedtotransformtherecipientC.

reinhardtii strainTN72via theglassbeadmethodasdescribed2.3.13.Figure3.22

illustratestheresultingC.reinhardtiicellline,TN72_SR_cpl1-HA_A_cpl1_c-HA.

Figure 3.22 TN72 recipient strain transformed with the pSRSapI_cpl1-HA_A_cpl1_c-HAplasmid,thusrescuingpsbHactivity.Carryingcpl1-HAunderthepsaA5’UTR,andcpl1_c-HAundertheatpA5’UTRinthechloroplast.

The resulting transformants were confirmed to be homoplasmic as described in

2.3.17andrepresentedinFigure3.23.Finally,correctinsertionintotheplastomewas

confirmed by PCR amplification from transformant genomic DNA and DNA

sequencing,asin2.3.18.

trnE2 psbH

TN72_SR_cpl1-HA_A_cpl1_c-HAtransformant MluI

5’psaA 1 cpl1-HA 3’rbcL5’atpAcpl1_c-HA3’rbcL

116

Figure 3.23 (A) Schematic describing the expected band when testing homoplasmictransformants.Thecombinationwhichproduces thenegativebandcanbeseen inFigure3.19. It istheabsenceofthenegative,350bp,bandthat indicateshomoplasmicity i.e.nocopies of the untransformed plastid remain (B) PCR products separated by gelelectrophoresisshowthecorrectexpectedlengthsandthusshowthatthenewtransformantisnowhomoplasmic.


InordertocomparetherelativeCpl-1accumulationlevelsbetweenTN72_SR_cpl1-

HAandTN72_SR_cpl1-HA_A_cpl1_c-HA,andinvestigatewhethertwocassetteswith

the samegenedoes indeed improveoverall proteinaccumulation levels in theC.

reinhardtiichloroplast,westernblotanalysiswasperformed.Wholecellextractsof

TN72_SR_cpl1-HA and TN72_SR_cpl1-HA_A_cpl1_c-HA were prepared, and five

technical replicates of each were separated by SDS-PAGE. After blotting to

3Kb

TN72

TN72_SR_

cpl1-HA

_A_cpl1_c-HA

1Kb

0.5Kb

psbH

TN72_A_cpl1_c-HAtransformant

MluIatpA.R psbH.R

2500bp

A

B

5’psaA 1 cpl1-HA 3’rbcL5’atpAcpl1_c-HA3’rbcL

atpA.R

psbH.R

117

nitrocellulose membrane as described in 2.4.5, the membrane was stained with

REVERT™WholeProteinStainwhichconfirmedsimilarquantitiesofcellpreparation

were loaded onto the gel (Appendix A) andwas used to normalize small loading

differences. After removal of the REVERT stain and blocking, themembranewas

probedwiththeanti-HAprimaryantibody,followedbyIRDye®secondaryantibody

andfluorescencewasobservedusingtheLiCorOdyssey®CLxsystem.Thewestern

blotimageandanalysisareshowninFigure3.24.

Figure3.24WesternblotanalysisenabledthequantificationofCpl-1accumulationinthetwoC. reinhardtii lines. Cpl-1 detection was normalised using the signal from REVERT TotalProteinStain.

Atwosamplet-testwascarriedouttocomparethelevelsofproteinaccumulationin

TN72_SR_cpl1-HA and TN72_SR_cpl1-HA_A_cpl1_c-HA, in order to identify any

statisticalsignificance.Thisisacomparisonofthemeanproteinaccumulationlevels,

0

0.1

0.2

0.3

0.4

0.5

0.6

WT1 WT2 WT3 WT4 WT5 2Cpl1R1

2Cpl1R2

2Cpl1R3

2Cpl1R4

2Cpl1R5

Relativedetectedfluoresence

***

TN72_SR_cpl1-HA TN72_SR_cpl1-HA_A_cpl1_c-HA

TN72_SR_cpl1-HATN72_SR_cpl1-HA_

A_cpl1_c-HA

Cpl-1(39kDa)

118

anditisatwo-tailedtest.Thenullhypotheses(H0)isthatthemeansarethesame.

Thealternativehypothesis(H1)isthatthemeansaredifferent.

H0:μ1=μ2

H1:μ1≠μ2

The full results can be seen in Figure 3.25, but in brief, there was a significant

difference in accumulation for TN72_SR_cpl1-HA (M = 0.247, Var = 0.001) and

TN72_SR_cpl1-HA_A_cpl1_c-HA(M=0.451,Var=0.007);t(8)=-5.14,p<0.001.

Figure3.25StatisticalreportgeneratedusingMicrosoftExceltocomparethelevelsofproteinaccumulationinthetwolinesofC.reinhardtii.

This result supports our original hypothesis - that protein accumulation can be

improvedintheC.reinhardtiichloroplastbyintroducingseveralcopiesoftheGOIas

differentexpressioncassettes.ItsuggeststhatCESislimitingtheleveloftranslation

foreachendogenous5’UTRandthatbyhavingthesamegeneunderthecontrolof

twodifferent5’UTRs,thiscanbeimproved.

t-Test:Two-SampleAssumingEqualVariancesTN72_A_cpl1-HA

TN72_SR_cpl1-HA_A_cpl1_c-HA

Mean 0.2472 0.4512Variance 0.0006 0.0073Observations 5 5PooledVariance 0.0039HypothesizedMeanDifference 0

df 8tStat -5.1423P(T<=t)one-tail 0.0004tCriticalone-tail 1.8595P(T<=t)two-tail 0.0009

tCriticaltwo-tail 2.3060

119

3.3.2.2.3 C.reinhardtiilineTN72_SR_cpl1-StrepII

Inthepreviousexperiment,itisnotpossibletodeterminehowtheproteinsynthesis

loadisbeingsharedbythetwoexpressioncassettes,astheproteinsarebothtagged

withanHAepitopeandarethereforeindistinguishableonawesternblot.

WethereforeproducedasimilarC.reinhardtiicelllinebutwithonecpl1encodinga

StrepIIepitopetaginsteadofanHAtag.ThisenablestheCpl-1proteinfromthetwo

different promoters/5’UTRs to be distinguished on a western blot and the

contributionofthetwocassettescompared.

ThefirststageinthisprocesswastoproducetheC.reinhardtiicelllineTN72_SR_cpl1-

StrepII,tobeusedasacontrolbutalsotoensurethatthistagcanbedetectedon

westernblotsandthattheproteinaccumulatestodetectablelevels.

3.3.2.2.3.1 Expressioncassetteproduction

Thefull-lengthcpl1-StrepIIgenewassynthesizedby IntegratedDNATechnologies.

SapI and SphI restriction enzymeswereused to clone the gene in-frame into the

pSRSapI expression vector, producing the plasmid pSRSapI_cpl1-StrepII. DH5α

competentE.colicellsweretransformedwiththeresultingplasmid.


ThepSRSapI_cpl1-StrepIIplasmidwasusedtotransformtherecipientC.reinhardtii

strainTN72viatheglassbeadmethodasdescribedin2.3.13.Figure3.26illustrates

theresultingC.reinhardtiicellline,TN72_SR_cpl1-StrepII.

Figure3.26TN72 recipient strain successfully transformed to carrycpl1-StrepII under thepsaA5’UTRinthechloroplast,andwithpsbHfunctionalityrestored.

trnE2 psbH

5’psaA 1 3’rbcL

TN72_SR_cpl1-StrepIItransformant MluI

cpl1-StrepII

120

The resulting transformants were confirmed for homoplasmicity as described in

2.3.17 (Appendix B). Finally, the sequence integrity of the inserted cassette was

confirmedbyDNAsequencingasin2.3.18.

3.3.2.2.3.3 Westernblotdetection

WholecellpreparationsofTN72_SR_cpl1-HA(asacontrol)andTN72_SR_cpl1-StrepII

were prepared and separated, in duplicate, by SDS-PAGE. After blotting to

nitrocellulosemembrane and blocking as described in 2.4.5, onemembranewas

probedwithananti-HAprimaryantibody,whilethesecondmembranewasprobed

withanti-StrepIIprimaryantibody.BothmembraneswerethenincubatedbyIRDye®

secondary antibody and fluorescencewasobservedusing the LiCorOdyssey®CLx

system.TheresultingimagesareshowninFigure3.27.

Figure 3.27 Two duplicatemembranes were probedwith anti-HA or anti-StrepII primaryantibodiesandCpl-1accumulationwasdetectedintheexpectedcelllinesi.e.Cpl-1-HAwasdetectedwiththeanti-HAantibodyandnotwiththeanti-StrepIIantibodyandviceversa.Thenon-specificbandseen in the lower imageconfirmsthatasimilarquantityofextractwasloadedontothegelforbothstrains.

3.3.2.2.4 C.reinhardtiilineTN72_SR_cpl1-StrepII_A_cpl1_c-HA

3.3.2.2.4.1 Transformationplasmidproduction

The plasmid was produced in the same manner as in 3.3.2.2.2.1, but using

pSRSapI_cpl1-StrepIIinsteadofpSRSapI_cpl1-HA.Theresultingplasmidwasnamed

pSRSapI_SR_cpl1-StrepII_A_cpl1_c-HA.

SR_Cpl1-HA

SR_Cpl1-StrepII

Cpl1(39kDa)

Anti-HA1ºantibody

Non-specific

Cpl1(39kDa)

Anti-strepII 1ºantibody

121


The pSRSapI_SR_cpl1-StrepII_A_cpl1_c-HA plasmid was used to transform the

recipientC.reinhardtiistrainTN72viatheglassbeadmethodasdescribedin2.3.13.

Figure3.28illustratestheresultingC.reinhardtiicellline,TN72_SR_cpl1-StrepII.

Figure3.28TN72 recipient strain successfully transformed to carrycpl1-StrepII under thepsaA 5’ UTR, and cpl1_c-HA under the atpA 5’ UTR in the chloroplast, and with psbHfunctionalityrestored.

Correct insertion of the two GOIs into the plastome and homoplasmy of the

transformantlineswereconfirmedasdescribedabove.


Inordertocompareaccumulationofproteinstaggedwithdifferentepitopetags,and

thereforedetectedviadifferentantibodies,aproteinstandardtaggedwithbothHA

and StrepII epitope tags was used to normalize the signal. This standard was

producedspecificallyforthispurpose,asdescribedin3.3.1.2.6.

C.reinhardtiiwholecellextractsfromthevariouscelllinesconstructedinthischapter

wereseparatedbySDS-PAGEinduplicate.Theproteinswereblottedtonitrocellulose

membranes as described in 2.4.5 and REVERT™ Total Protein stain was used to

normalise loading error (Appendix C). One membrane was probed with anti-HA

primaryantibody,whilethesecondmembranewasprobedwithanti-StrepIIprimary

antibody.Bothmembraneswere then incubatedwith IRDye® secondaryantibody

andfluorescencewasobservedusingtheLiCorOdyssey®CLxsystem.Theresulting

imagesareshowninFigure3.29.

trnE2 psbH

TN72_SR_cpl1-StrepII_A_cpl1_c-HAtransformant MluI

5’psaA 1 3’rbcL5’atpAcpl1_c-HA3’rbcL cpl1-StrepII

122

Theproteinstandard,whichistaggedwithbothHAandStrepIIepitopetagswasthen

usedtonormalizethetwomembranes,aswastheREVERT™totalproteinstain.The

relativeproteinaccumulationlevelsofeachC.reinhardtiistrainwerecomparedin

Figure3.29(B).TN72_SR_cpl1-HA,whichhasbeenusedthroughoutthischapterasa

benchmarkforCpl-1proteinaccumulationlevel,uponwhichtoimprove,wassetto

100%andotherlevelsarerepresentedaspercentagesofthis.

123

Figure3.29(A)WesternblotanalysisofC.reinhardtiiwholecellextractsprobedwithanti-HAoranti-StrepIIprimaryantibodies.IRdetectionwasperformedbytheLiCorOdyssey®CLx.(B)ProteinaccumulationlevelswerequantifiedusingtheOdyssey®ImageStudiov5.0andthesignalwasnormalisedusingtheproteinstandardandREVERT™totalproteinstaining.TN72_SR_cpl1-HAisthebenchmarkstraininthisinvestigationandwasthereforesetto100%accumulation.Other strains are represented relative to this. Protein accumulation of theStrepIItaggedCpl-1appearstobeconsiderablylowerthanthatoftheHAtaggedprotein.

As observed in Figure 3.24, the level of Cpl-1 protein accumulation in the

TN72_SR_cpl1-HA_A_cpl1_c-HAisapproximatelyequaltothesumofTN72_SR_cpl1-

HAandTN72_A_cpl1_c-HA.However,itisinterestingtonotethataccumulationof

StrepII-taggedCpl-1isconsiderablylowerthanitsHA-taggedequivalent.Thiscould

SR_cpl1-HA

SR_cpl1-StrepII

Cpl-1

Anti-HA1ºantibody

Non-specific

Cpl-1

Anti-strepII1ºantibody

A_cpl1_c-HA

SR_cpl1-HA

_A_

cpl1_c-HA

SR_cpl1-StrepII_

A_cpl1_c-HA

SR_Contro

l

Standard(10ug/ml)

0%

50%

100%

150%

200%

250%

300%

SR_cpl1-HA

SR_cpl1-StrepII

A_cpl1_c-HA

SR_cpl1-HA

_A_

cpl1_c-HA

SR_cpl1-StrepII_

A_cpl1_c-HA

SR_Contro

l

Anti-HA Anti-StrepII

A

B

124

beduetoeithertheStrepIItagdestabilizingtheprotein,oritcouldpossiblybean

artefactofthenormalizationmethod.

Theadditionofanypeptidetoaproteinhasthepotentialtodestabiliseitbyaffecting

theabilityorrateoffoldingoftheproteinintoastableform,andthismaybethe

casewiththeStrepIItaggedCpl-1.

Alternatively,itispossiblethattheStrepIItagontheproteinstandardismoreeasily

accessiblethanthatonCpl-1,orconverselythattheHAtagislessaccessibleonthe

standardthanonCpl-1.Thisscenariowould leadtoanunderestimationofStrepII

taggedproteins,oranoverestimationofHAtaggedproteins,respectively.Itseems

plausible that a little of both scenariosmay be occurring, given that the protein

standardistaggedwithanHAtagattheC-terminalend,followedbytheStrepIItag

beyondthat.Anti-HAantibodiescouldthereforebeslightlyhinderedbythepresence

of the StrepII tag, while anti-StrepII antibodiesmay find the epitope to bemore

accessibleduetoitsslightlyincreasedprotuberancefromtheCpl-1protein.

To investigatewhich of these theories is correct, two separate protein standards

couldbecreatedforeachtaggedCpl-1.IfmuchlowerlevelsofStrepIItaggedCpl-1

arestillobserved,thenitcouldbeconcludedthattheStrepIItagisdestabilizingCpl-

1,butifaccumulationlevelsappeartobesimilarthenitwasmerelyanartefactof

thenormalizationprocess.

Ineithercase,itappearsthattheinsertionofmultipleexpressioncassettesintothe

C.reinhardtiichloroplastresultsinasimpleadditionoftheproteinlevelsobserved

whencassettesareinsertedsingly.

3.3.3 Codonpairoptimizationforimprovementofproteinaccumulationlevels

The second aspect of this chapter looks at using codon pair optimization as a

techniqueforimprovingrecombinantproteinaccumulationlevelsintheC.reinhardtii

chloroplast.Codonoptimizationisawell-knownapproachtoimproverecombinant

protein yields, and has been shown to be particularly important for transgene

expression in theC. reinhardtii chloroplast – Franklin et al. (2002b) increasedgfp

125

expression by 80-fold in a codon optimized version compared to a unoptimized

version. The codon frequency chart for theC. reinhardtii chloroplast is shown in

AppendixD.Taunt(2013) investigatedtherolethatcodonpairsplayandwhether

these too couldbeoptimized to improveexpression. The study identified several

codonpairswhichhadhighpredictedoccurrencebutwererarelyorneverobserved

in the C. reinhardtii chloroplast plastome. These pairs were terms Unexpectedly

Unseen Zero Scoring Codon Pairs (uuZSCP). Two such uuZSCP stood out as being

particularlysignificant,aPhenylalanine:Argininepair,andanAsparticAcid:Leucine

pair, shown in Figure 3.30. The Phe:Arg pair was chosen for investigation in this

projectasithappenedtobeincludedintheinitialcodonoptimizationforcpl1,andis

thereforepresentinTN72_SR_cpl1-HA.

Figure3.30TwouuZSCPareparticularlyrareintheC.reinhardtiichloroplast,despitetheirconstitutivecodonsbeingrelativelycommon.Therelativefrequencyreferstothefrequencywithwhich this codonwouldbechosen to represent thisaminoacid in theC. reinhardtiichloroplast.ThePredictedOccurrenceishowoften,giventhefrequencyofaminoacidpairs,onewouldexpecttoseethiscodonpairinthetotal2579codonpairsintheC.reinhardtiichloroplast.

FirstCodon SecondCodon Codon PairAminoAcidPair

Code RelativeFreq. Code Relative

Freq.

CombinedRelativeFreq.

PredictedOccurrence

ActualOccurrence

Phe:Arg UUU 0.67 CGU 0.73 0.49 28.29 0Asp:Leu GAU 0.76 CUU 0.14 0.1 9.1 0

126

Figure3.31TheaminoacidsequenceofCpl-1.ThePhe:Argpairishighlightedinyellow,whilethepurpleboxindicatestheHAtag.

AccordingtoMauletal(2002),theC.reinhardtiichloroplastplastomecontains30

identifiedtRNAgenes,providingasmallyetsufficientsetforproteinsynthesis.After

examination of the data and checking the chloroplast genome (GenBank:

BK000554.2) for tRNA genes using tRNAscan-SE (http://lowelab.ucsc.edu/cgi-

bin/tRNAscan-SE),acoupleofinaccuraciesinthetRNAdatawerenoticed.Firstly,the

trnEgeneiscountedthreetimes,wheninfactthereappeartobeonlytwocopies

identified on the plastome. There are therefore only 29 tRNA genes in the C.

reinhardtiichloroplast.Secondly,thetrnR2islabelledwiththeincorrectanti-codon

–itshouldreadtrnR2(UCU),nottrnR2(AGA)–likelyjustaconfusionbetweencodon

andanticodon.Thirdly,whilenotamistakeperse,thenomenclatureisunclearfor

tRNAgenesand seems tobe inconsistent. Forexample, thereare trnR and trnR2

genes,whichidentifytRNAswithdifferentanticodons,butnotrnR1exists.Atableof

allthetRNAgenesintheC.reinhardtiichloroplasthasbeencompiledinFigure3.33.

TheC. reinhardtii chloroplast chromosomemapbyMauletal. (2002) is shown in

MVKKNDLFVDVSSHNGYDITGILEQMGTTNTIIKISEST

TYLNPCLSAQVEQSNPIGFYHFARFGGDVAEAEREAQFF

LDNVPMQVKYLVLDYEDDPSGDAQANTNACLRFMQMIAD

AGYKPIYYSYKPFTHDNVDYQQILAQFPNSLWIAGYGLN

DGTANFEYFPSMDGIRWWQYSSNPFDKNIVLLDDEEDDK

PKTAGTWKQDSKGWWFRRNNGSFPYNKWEKIGGVWYYFD

SKGYCLTSEWLKDNEKWYYLKDNGAMATGWVLVGSEWYY

MDDSGAMVTGWVKYKNNWYYMTNERGNMVSNEFIKSGKG

WYFMNTNGELADNPSFTKEPDGLITVAYPYDVPDYA

127

Figure3.34.ItshowsthelocationofallthetRNAgenesontheplastomeandexplains

thatthetwocopiesoftrnAandtrnIareduetotheirlocationontheinvertedrepeat

sequences.

InordertotestwhetherthepresenceoftheuuZSCPPhe:Argcodonpairisaffecting

thelevelofproteinaccumulationinTN72_SR_cpl1-HA,twocpl1genemutantswere

createdbysite-directedmutagenesis.Thefirst,CPOCv1(CodonPairOptimizedCpl-1

version1),wasamutationofthephenylalaninecodonfromUUUtoUUC–thereis

onlyonetrnFgene,sothiscodonwouldbeservedbythesametRNA“wobbling”as

cpl1.Thesecondmutant,CPOCv2, includesamutationoftheargininecodonfrom

CGUtoAGG–thiswasdesignedtobeservedbythetRNAencodedbytrnR2,rather

thanthatencodedbytrnR1whichservescpl1.

N.B.AGAisaslightlymorefrequentlyseenArgininecodonthanAGG,asshownin

Figure7.4,andwouldnotrelyontRNAwobble.However,UUU:AGAisalsoacodon

pairthatisneverobservedinthechloroplast,soforthisreason,UUU:AGGwhichis

observed,waschosen.

Phe Arg CPAI Presentin…UUU CGU 0 cpl1

UUU CGC 0 UUU CGA 0 UUU CGG 0 UUU AGA 0 UUU AGG 0.0625 cpov2

UUC CGU 1 cpov1

UUC CGC 0 UUC CGA 0.0625 UUC CGG 0 UUC AGA 0.0625 UUC AGG 0

Figure3.32AllpossiblecodoncombinationsforaPhe:Argpair.Thecodonpairadaptationindex(CPAI)istheratioofcodonpairfrequencytothefrequencyofthemostobservedcodonpair.Thedataabovearefroma“hand-pick”databaseintheCodonUsageOptimizerBeta

0.92 (Kong 2013) software, whereby the codon pair usage in highly expressed genes iscollected.

128

Giventhetheoryforcodonpairbiaslaidoutin3.1.4,itwashypothesisedthatCPOCv1

wouldbeunaffectedby thecodonchange,whileCPOCv2maydisplayadifferent,

possiblyhigher,levelofCpl-1proteinaccumulation.

Figure3.33A listoftRNAgenes identifiedbyMauletal. (2002).TheyellowrowsindicateduplicatetRNAgenes,i.e.theyhavethesameanticodon.GreenrowsindicatetRNAgeneswithdifferentanti-codons,butcodingforthesameaminoacid.

No. Name Anticodon Codon AminoAcid

1trnA UGC

GCA AlatrnA UGC

2 trnC GCA UGC Cys3 trnD GUC GAC Asp

4trnE UUC

GAA GlutrnE UUC

5 trnF GAA UUC Phe6 trnG1 UCC GGA Gly7 trnG2 GCC GGC Gly8 trnH GUG CAC His

9trnI GAU

AUC IletrnI GAU

10 trnK UUU AAA Lys11 trnL1 UAG CUA Leu12 trnL2 UAA UUA Leu13 trnM1 CAU AUG fMet14 trnM2 CAU AUG Met15 trnM3 LAU AUA Ile16 trnN GUU AAC Asn17 trnP UGG CCA Pro18 trnQ UUG CAA Gln19 trnR1 ACG CGU Arg20 trnR2 UCU AGA Arg21 trnS1 UGA UCA Ser22 trnS2 GCU AGC Ser23 trnT UGU ACA Thr24 trnV UAC GUA Val25 trnW CCA UGG Trp26 trnY GUA UAC Tyr

Phenylalanine

Arginine

129

Several naming changes have been made in Figure 3.33 to the original names

publishedinMauletal.(2002).Thenewnamesfallinlinewiththeannotationsmade

to the chloroplast genome available at NCBI

(https://www.ncbi.nlm.nih.gov/nuccore/BK000554).Thenamesmeanthatidentical,

duplicategenes (trnA, trnIand trnE)have thesameanticodon,whilegeneswhich

encode similarly charged tRNAs,butwithdifferinganticodons, arenumberede.g.

trnL1andtrnL2.TheonlychangemadefromtheNCBInamingistonamebothtrnE

genesthesame,astheyareidenticalduplicates.Itshouldalsobenotedthatsome

discrepancyexistsbetween thedefinedgene lengthonNCBIand the tRNAgenes

detectedusing the tRNAscan software,most likelydue to incorrect annotationof

NCBI.

130

FigureRemoved

Figure 3.34 TheC. reinhardtii chloroplast chromosome. tRNA genes are identified on theouterringinblackandlabelledwithasingleletter.e.g.V,whichindicatestrnV.TwotRNAgenes (trnA and trnI) are located in the Inverted Repeat (IR) regions of the plastome,indicatedinthickblack,thusexplainingtheirduplication.ThesecondcopyoftrnL(2o’clock)shouldbeblacktoindicatetRNA,asshouldtrnIandtrnAontheright-handIR.Reproducedfrom Maul et al. (2002). Note: As an aside, the neutral site used for chloroplasttransformationthroughoutthisthesisislocatedbetweentrnEandpsbH,5o’clock.

131

3.3.3.1 CodonPairOptimizedCpl-1version1and2(CPOCv1andCPOCv2)

CPOCv1wasdesignedsuchthatadifferentcodoncodedforphenylalanine,butthat

codonwouldstillbeservedwiththesametRNAaswasusedintheoriginal,through

the phenomenon known as tRNA wobble (Alkatib et al. 2012). CPOCv1 would

thereforeactasacontrol for this investigation,as the theorybeing tested is that

tRNAsinteractintheAandthePsiteoftheribosomeinamannerthatcaninfluence

translationefficiency.Here, the same tRNAswill beused in thismutant as in the

originalversionandthusweexpecttoseenochangeintranslationefficiency.

CPOCv2wasdesignedsuchthatadifferentcodoncodedforarginine,andthatthat

codonwouldbeservedwithadifferenttRNAtotheoriginal,astherearetwotrnR

genes in the C. reinhardtii chloroplast with different anti-codons. CPOCv2 was

producedinanattempttoinfluenceaccumulationlevelsofCpl-1,asdifferenttRNAs

willbeinteractingintheAandthePsiteoftheribosomecomparedtothoseinthe

originalversion,andthereforetranslationmayprogressatafasterrate.

3.3.3.1.1 Codonchangebysite-directedmutagenesis

TocreateCPOCv1,primersweredesignedtomutateU→Cinthethirdpositionof

thephenylalaninecodonthroughPCR,andforCPOv2tomutateC→Ainthefirst

positionandT→Ginthethirdpositionoftheargininecodon.Thestepstakento

performthismutagenesisareshowninFigure3.35andFigure3.36respectively.The

primersusedareindicatedbelow.Thelowercaselettersshowthesitesformutation.

cpl1.F: GCTCTTCAATGGTTAAAAAAAATGATTTATTC

cpocv1_SDM.F: AGGTGGTGGTTcCGTCGTAA

cpocv1_SDM.R: TTACGACGgAACCACCAACCT

cpocv2_SDM.F: GTTGGTGGTTTaGgCGTAATAATG

cpocv2_SDM.R: CATTATTACGcCtAAACCACCAAC

cpl1.R: GCATGCTTATTAAGCATAATCTGGAAC

132

Figure3.35Site-directedmutagenesiswasperformedasabovetomodifythephenylalaninecodontomakeCPOCv1.PrimersareshowninblueandPCRconditionsaredescribedin2.3.7.

Figure3.36Site-directedmutagenesiswasperformedasabovetomodifytheargininecodontomakeCPOCv2.PrimersareshowninblueandPCRconditionsaredescribedin2.3.7.

3.3.3.1.2 Expressioncassetteproduction

The expression cassette was assembled as with previous examples. The gene,

includingtheHAtagattheCterminus,wasclonedintothepSRSapIvectorinframe

usingSapIandSphIrestrictionsites.TheinsertionwasconfirmedbyDNAsequencing

TTT CGTAAA GCA

AAG GCA

TTC CGT

TTC CGTAAG GCA

TTC CGTAAG GCA

1st PCR

2nd PCR

Finalproduct

Cpl-1

CPOCv1

cpl1.F

cpl1.R

cpocv1_SMD.F

cpocv1_SMD.R

TTT CGTAAA GCA

AAA TCC

TTT AGG

TTT AGGAAA TCC

TTT AGGAAA TCC

1st PCR

2nd PCR

Finalproduct

Cpl-1

CPOCv2

cpl1.F

cpl1.R

cpocv2_SMD.F

cpocv2_SMD.R

133

of the plasmid as described in 2.3.18. The resulting plasmids were called

pSRapI_CPOCv1-HAandpSRapI_CPOCv2-HA.

3.3.3.1.3 C.reinhardtiitransformation

Two TN72 transformations were performed, using pSRSapI_CPOCv1-HA and

pSRSapI_CPOCv2-HA plasmids. Transformed C. reinhardtii lines were checked for

homoplasmicitybyPCRandfinallyconfirmedbysequencing.

3.3.3.2 Western blot quantification of TN72_SR_CPOCv1-HA and

TN72_SR_CPOCv2-HA

Itishypothesisedthatthecodonchangesmadeabovewillresultindifferentlevels

ofproteinaccumulationbetween the two transformant lines.To test this, fourC.

reinhardtiicultures,TN72_SR_cpl1-HA,TN72_SR_CPOCv1-HA,TN72_SR_CPOCv2-HA

and TN72_SR_Control, were grown in an Algem Photobioreactor simultaneously,

withthesamestartingcelldensityandwiththesamephysiologicalconditions.OD740

wasmeasuredautomaticallyevery30minutesandsamplesweretakenatintervals

foranalysisbywesternblotting.1mlsampleswerecollectedfromtheculturevolume

andimmediatelysnap-frozeninliquidnitrogenandstoredat-80°C.Theculturewas

grownfor145hoursintotal.Attheendofthistimethefrozensampleswereanalysed

togetherbywesternblotting.Whole-cellextractswereseparatedbySDS-PAGE,with

nocompensationmadeforcelldensity.Afterblottingtonitrocellulosemembrane

andblocking,themembraneswereprobedwithanti-HAprimaryantibody,followed

by IRDye® secondary antibody and fluorescence was observed using the LiCor

Odyssey®CLxsystem.

Figure3.37showsthewesternblotanalysis–ariseinproteinaccumulationovertime

ascelldensityincreasesistobeexpected.

134

Figure3.37WesternblotanalysisoffourC.reinhardtiistrainsatfourtimepoints.Theknownstandardwasusedtonormaliseanydifferencebetweenblottingonseparatemembranes.

The quantified and normalised data is displayed in Figure 3.38, alongside optical

density data collected by theAlgemphotobioreactor. Thewhole experimentwas

repeatedasecondtime,andthefinalresultscanbeseeninFigure3.39,withwestern

blotanalysisinAppendixE.

3.3.3.2.1 Statisticalanalysisofwesternblotdata

A one-way ANOVA test was carried out on the recombinant protein yields of

TN72_SR_cpl1-HA, TN72_SR_CPOCv1-HA and TN72_SR_CPOCv2-HA in order to

identify any statistical difference between the levels of protein accumulation at

differenttimepoints,detailedinAppendixF.Inthefirstrunoftheexperiment,itwas

foundthattheproteinaccumulationat45hoursand58hourswassignificantlyhigher

inTN72_SR_CPOCv2-HAthanintheothercelllines,butthattherewasnosignificant

difference beyond this point.On the other hand, the second performance of the

experimentwasrunforlongerandlikewise,suggeststhatproteinaccumulationof

TN72_SR_CPOCv2issignificantlyhigherthe45and56-hourtimepoints.However,

CPOCv1 and CPOCv2 then continue to rise, while the protein accumulation in

TN72_SR_cpl1-HArisesatamuchlowerrate,beingsignificantlylowerthanthecodon

pair optimised versions at the 112, 139 and 194-hour time points. The statistical

methodandresultsaredetailedinAppendixG.

TN72_SR_cpl1-HA

45h

Standard

(10ug/ml)

TN72_SR_CPOCv1-HA

TN72_SR_Control

TN72_SR_CPOCv2-HA

58h

77h

119h

135

Figure3.38Algemgrowthdata(linechart,lefty-axis)andwesternblotquantification(barchart,righty-axis).TheAlgemphotobioreactorrecordedopticaldensityat740nmevery90seconds.AtOD740=~2.5,thephotobioreactorisunabletorecordanyhigherODduetothelongpath-lengthacrossa400mlculture.However,asindicatedbytheproteinaccumulationlevels,theculturesarereachingtheirstationaryphasearoundthispointanyway.TherelativedetectedfluorescenceforHA-taggedproteinaccumulationistakenfromFigure3.37,andnormalisedusingtheproteinstandardandtheOD740.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0

0.5

1

1.5

2

2.5

3

0 12 24 36 48 60 72 84 96 108 120 132 144

Relativ

edetectedflu

oresence


)

Time(hours)SR_cpl1-HAProtein SR_CPOCv1-HAProtein SR_CPOCv2-HAProtein SR_ControlProteinSR_Cpl1-HA SR_CPOCv1-HA SR_CPOCv2-HA SR_Control

****

****

136

Figure3.39Algemgrowthdata(linechart,lefty-axis)andwesternblotquantification(barchart,righty-axis).ThestartingODwaslowerinthisexperiment,sotheculturetakeslongertoreachthelogarithmicgrowthstage.TheoriginalwesternblotanalysisisshowninAppendixE,inthisinstancethe25kDabandoftheproteinladderwasusedtonormalisethemembranes,thisenabledmoresamplestobefittedontoasinglegelandthusmeasureaccumulationoveralongertimeperiod.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0

0.5

1

1.5

2

2.5

3

0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192

Relativ

edetectedflu

oresence


)

Time(hours)

SR_cpl1-HAProtein SR_CPOCv1-HAProtein SR_CPOCv2-HAProtein SR_ControlProteinSR_Cpl1-HA SR_CPOCv1-HA SR_CPOCv2-HA SR_Control

** **

* *

*

137

Figure3.38indicatesthatCPOCv1isgrowingslightlyslowerthanthetwocontrols,

andCPOCv2isgrowingslightlyfaster.This ismost likelyduetoaslightlydifferent

numberofcellsusedtoinoculatethecultures,despiteeffortstoensuretheywere

the same. However, to compensate for thiswhen comparing protein yields from

westernblotanalysis,thedetectedsignalwasdividedbytheOD,thusnormalising

thedataforcelldensity.Eachofthefourmembranesusedforwesternblotanalysis

alsoshowedslightlydifferentsignalsfortheproteinstandard,probablyduetonon-

uniformtransferfromthegeltothemembrane,slightlydifferentantibodyincubation

times and slightly different dilutions of milk and antibody. Assuming that these

variables are the same within membranes, but can vary from membrane to

membrane, the datawere normalised by division of the average level of protein

standarddetected.ThesameistrueforFigure3.39,butthistimeusingthe25kDa

bandoftheproteinladdertonormaliseforinter-membranedifferences,astherewas

noroomonthemembranetoincludetheproteinstandardtoo.

Whilethetwographscannotbecompareddirectly,duetothelongerlagtimeinthe

latter and different inter-membrane normalisation techniques, they do appear to

displaytwotrends.Firstly,thatCPOCv2seemstoaccumulatefasterthantheother

Cpl-1variantsthroughoutthe logarithmicgrowthstage,andsecondlythattheun-

optimisedversionofCpl-1consistentlyaccumulatestoalowerlevelthaneitherof

theCPOversions.

This is an interesting observation, as according to the theory of codon pair

optimisation,CPOCv1accumulationshouldbenodifferenttotheunchangedCpl-1,

as the tRNAs serving the mRNA are identical. However, particularly in the later

growth stages in Figure 3.39, there is a consistently and significantly higher

accumulationofCPOCv1andCPOCv2.

Given the evidence from section 3.3.1, in which datawas collected on the error

introducedatvariouspointsofmeasuringproteinaccumulation,thesedifferences

seem too large to be artefacts of variability in western blotting and cell density

measuring techniques. Therefore, alternative explanations for this resultmust be

sought.

138

The first thing to consider is the fact that in CPOv2, another codon pair is being

unintentionallyaltered.Theaminoacidimmediatelyfollowingthecodonoptimized

ArginineisanotherArginine,ascanbeseeninFigure3.31.ThisArg:Argcodonpairis

beingunintentionallyalteredwhentheprecedingPhe:Argcodonpairisoptimizedin

CPOv2.Thispresentsanothervariablethathasnotbeenaccountedfor,andasshown

in Figure 3.40, the new pair is never observed in the “hand-picked” database,

consistingofhighlyexpressedproteinsintheC.reinhardtiichloroplast,generatedby

Taunt(2013).Thispresentsanissue,asalthoughoneuuZSCP(UUU:CGU)hasbeen

replaced,anotherunseenpairisintroduced(AGG:CGU).Itis,therefore,possiblethat

thebeneficialeffectsof the formerarenegatedby thedetrimentaleffectsof the

latter. There is no similar effectmade by CPOv1, as all Phenylalanine codons are

servedbyasingletRNA.

Arg Arg CAI Presentin…CGU CGU 1 cpl1&cpov1CGC CGU 0 CGA CGU 0 CGG CGU 0 AGG CGU 0 AGA CGU 0 cpov2

Figure3.40Datatakenfromthe“hand-pick”databaseofCodonUsageOptimizerBeta0.92(Kong 2013). This is a database of codon usage in highly expressed proteins, a commonmethodofselectingthebestcodonusage.

Anotherpossibilityisthatwhilegeneexpressionappearstobeprimarilycontrolled

attheleveloftranslationintheC.reinhardtiichloroplast,thetranscriptionallevelstill

mayplayapartandassuchthelevelsofmRNAproducedforCPOCv1andCPOCv2

shouldbeinvestigated.Ifthesearefoundtoremainsimilarthenitcanbediscounted,

butshouldbeinvestigatednevertheless.

Additionally, the unexpectedly unseen nature of UUU:CGU could be just a

coincidence.Althoughthisseemedunlikely,giventheconvincingstatisticalanalysis

performedby Taunt (2013), the assertion that this pair is purposefully avoided is

slightlyunderminedbythefrequentpresenceofUUC:CGU.UUC:CGUaccountsfor33

ofthe59observedinstancesofPhe:ArgintheC.reinhardtiichloroplast,butgiven

139

thefactthatUUUandUUCareservedbythesametRNA,thedifferentcodonshould

confer no difference in translational efficiency or codon selection. It could be

thereforethatsomeneutralmechanismhasresultedinthisbias,notselection,for

example, preferentialmutationbias duringDNA replicationor repair (Lucks et al.

2008).Anotheralternativeisthatthesinglebasechangeinthecpl1transgenehas

created an unexpected local area of secondary structure within the mRNA, thus

altering the translationalefficiency (Gasparetal.2013).Thiskindof interaction is

very difficult to predict, as the mRNA folding events may involve two distant

sequences,andchangingonebasemightstrengthen/weakentheseinteractions.

Thefinalandpossiblymostlikelyexplanationisonewhichislittleunderstood.Thisis

thefrequentlyobservedandreportedphenomenonofvariabletransgeneexpression

between“identical”C. reinhardtiichloroplast transformants (MayfieldandSchultz

2004; Surzycki et al. 2009; Michelet et al. 2011). The differences can be quite

remarkable: Surzycki et al. (2009) reported that recombinant protein expression

levelsofVP28,aWhiteSpotSyndromevirusprotein,variedbetweentransformants

from0.88%to20.9%TCP.Whenonlyalownumberoftransformantsaregenerated,

as is frequently thecasewiththeglassbeadmethodregularlyused in thePurton

group, a significant difference in transgene expression could be incorrectly

apportioned to a factor which really has little effect. The reason put forward by

Surzycki et al. (2009) for this observation is transformation-associated genotypic

modifications,termedthe“transformosome”.Thepaperpostulatesthatduringthe

process of genetic transformation, fragments of the transformation plasmid are

unknowingly inserted into theplastomeor nuclear genome, thus alteringnuclear

genes that regulate chloroplast gene expression, protease expression, or sites of

regulatory gene action in the chloroplast. Evidence is presented that the level of

steadystatetransgenemRNAisunaffected,givingfurtherweighttothebeliefthat

generegulationoccursprimarilyatthetranslationlevelinC.reinhardtii.

C. reinhardtii is extremely good at resourcemanagement, and as such is able to

prioritise theproductionofkeyproteinsoverothers, forexamplewhenCopper is

limitingthecellwillpreferentiallyproducecytochromeCoxidaseoverplastocyanin

foruseintheelectrontransferchainofPS1(MerchantandBogorad1986).However,

140

asCpl-1isarecombinantprotein,itisnotinvolvedinthishierarchyandistherefore

likelytobethefirstproteintobesacrificedinthecaseofametaboliclimitation.As

the value for protein accumulation is relative (i.e. it has been normalised for the

opticaldensityoftheculture),Figures3.38and3.39bothshowincreasinglevelsof

protein accumulation as the culture progresses into stationary phase and then it

plateaus. Therefore, metabolic limitation does not appear to be an issue in this

instance.

Toconclude,itappearsthatadifferenceinCpl-1accumulationhasbeenobserved,

butitisunclearastowhetherthisdifferenceisduetothecodonpairoptimisation,

astheresultsdonotsupportthetheorythatcodonpairscanbeoptimisedduetothe

differentinteractionsoftRNAsattheAandP-sitesoftheribosome,andtheobserved

differencesaresmallenoughtobeaccountedforbyotherfactors.

3.4 Conclusionandfuturework

This chapter has investigated two putative methods of improving recombinant

protein expression in the C. reinhardtii chloroplast: the introduction of multiple

expression cassettes; and the application of codon pair optimisation. The former

appearstobeamethodtorobustlyimproveproteinaccumulation,whilethelatter

method remains unclear as the complex and incremental nature of the change

appearstobemaskedbyotherfactorsinplay.Interestinglytheconcernaddressed

atthestartofthechapter,namelyhowtoaccuratelycompareproteinaccumulation

between transformant lines, re-emerged as an issue towards the end of the

investigation.

3.4.1 Multipleexpressioncassettestrategy

TheoverallconclusionfromthisstudywasthatC.reinhardtiilinestransformedwith

multiple expression cassettes produce a recombinant protein yield approximately

equal to the sum of the yields observed in lines transformedwith each cassette

individually.ThisfindingcouldpotentiallybeusedtodoubleexistingyieldsintheC.

reinhardtiichloroplast.However,somefurtherworkintowhatthelimitationsofthis

methodarewillenableustobetterunderstanditspotential.

141

Thefirstandmostobvioustestistoinvestigatewhetherthisresultcanberepeated

withothertransgenes.Thiswouldbearelativelystraightforwardtesttoperform,but

itwouldbeinterestingtotestbothanotherhighlyexpressedgene,aswellasagene

whichiscurrentlyonlyweaklyexpressedintheC.reinhardtiichloroplast.Thecpl1

geneiswellexpressed,andthereforeitispossiblethatCESmechanismsarelimiting

its accumulation under a single 5’ UTR, thusmaking it a good candidate for this

technique. On the other hand, proteins displaying poor accumulation might be

limitedbyproteaseactivityforexample,andwillthereforeremainatlowlevelsof

accumulationdespitedifferentmRNAtranscripts.

Thesecondquestionthatonewouldliketoaskistowhatpointcanthistheorybe

extended.Willthreeorfourcassettestripleorquadrupletheproteinaccumulation,

for instance? Going beyond two copies of the gene raises the issue discussed in

section3.3.2.2–whentwoduplicategenesareinparalleldirectionstoeachother,

thereisapotentialforDNAexcisiontooccurbetweentherepeats.Avoidingthisby

increasingthedistancebetweenthecopieswouldmostlikelyrequireanotherneutral

site to be used and targeted for transformation, requiring the use of a second

selectablemarker, enabling sequential transformations. Two options exist in this

case, either the addition of a positive selectable marker, such as aadA and

maintaining the algae on spectinomycin containing media, or the addition of a

negativeselectablemarker,suchascodA,totherecipientlinewhichisthenreplaced

inthesecondtransformationevent(YoungandPurton2014).Thelatterwouldresult

in the formation of a marker-less mutant which is desirable for further

transformationsbutrequiresmoretimetomaketheconstructs.Asaninitialtestof

how far themultiple-cassette theory can be taken, it is suggested that using an

antibiotic resistancemarker ismorestraightforward.Furthermore, thepracticeof

usingcodonmanipulationtominimisethepotentialforDNAexcision,asperformed

in3.3.2.2.1.1,mayenablemorecassettestobeinsertedintothesamesite.However,

given that codon optimisation has been shown to be very influential in protein

accumulation theC. reinhardtii chloroplast (Surzycki et al. 2009), this canonlybe

takensofarwithoutaconsiderablereductionintranslation.

142

Asdiscussedin3.3.2.2.4.3,aninvestigationintowhytheStrepIIepitopeappearsto

beaffectingproteinaccumulationsoprofoundlyshouldbeperformed.Thesimplest

explanation is that normalising to the protein standard is skewing the results by

havingadifferentepitopeaccessibilitythanthatofthesinglytaggedproteins.This

wasanissueencounteredpreviouslyinthePurtongroup(Taunt2013;Stoffels2015),

wherebythecommercialmulti-taggedproteinstandardsuggestedlevelsofprotein

accumulationthatcouldnotbeconfirmedbySDS-PAGEandCoomassieBluestaining.

Furthermore,asimilarresultin5.3.2.4.3suggeststhatthisisthecase,whereanother

endolysin,CD27L,alsoappears toaccumulate toamuch lower levelwhenStrepII

tagged.However,itmaybethattheendolysinisextremelysensitivetoC-terminal

epitopetagsintermsofstability,inwhichcaseaninvestigationintotheaccumulation

usingdifferentepitopetagscouldbecarriedoutandoneshouldalsoconsidertagging

theN-terminusinstead.

Finally,acalculationofrecombinantproteinyieldasapercentageofTotalSoluble

Protein(TSP)orTotalCellProtein(TCP)wouldbepossibleoncetheissuesurrounding

theproteinstandardhasbeenresolved.Thiswouldallowcomparisontootheryields

achieved in the literature. Alternatively, an ELISA assay could be performed but

antibodies would have to be raised against Cpl-1, which is expensive. A robust

methodofestimatingproteinyieldusinganepitopetagandwesternblotanalysis

wouldbecheaperandmoreflexible,enablingquantificationofotherproteinsinthe

future.

3.4.2 Identicaltransformanttransgenevariability

Theissueoflargevariabilityintransgeneexpressionbetweensupposedlyidentical

transformants, also known as “clonal variation”, has the potential to mask or

exaggerate investigations into othermethods of improving protein accumulation.

Untilthisissuehasbeenaddressed,“fine-tuning”techniqueswillremaindifficultto

assayandareunlikelytobeaseffectiveassimplyscreeningmanytransformantsand

selecting the best, as many protein production companies working with Pichia

pastoriscurrentlydo(Awetal.2017).

143

The most useful tool to aid investigation into this phenomenon would be the

development of a sensitive and robust reporter gene for C. reinhardtii. Reporter

genes such asGFP have beenused for this purpose inmany other organisms, as

fluorescence is graded and easily measured in vivo, enabling rapid quantitative

screening of many transformants. However, while some studies have produced

reasonablelevelsofGFPintheC.reinhardtiichloroplast(Franklinetal.2002),dueto

thehighlevelsofauto-fluorescenceinC.reinhardtii,GFPisunsuitableasitmustbe

producedathighlevelstobedetectable(MayfieldandSchultz2004).Luciferasehas

beendevelopedasaviablealternative,withfluorescenceshowntobeproportional

toproteinaccumulation,andnointerferencebycellauto-fluorescence(Mayfieldand

Schultz 2004). Surzycki et al. (2009) demonstrated that co-introduced selectable

markergenesareexpressedinamannerproportionaltotheGOI.Therefore,theco-

introductionofluciferasewithaGOIshouldenableitsfluorescencetobeusedasa

reliable proxy for recombinant protein accumulation. This in turnwill enable the

rapidscreeningofmanydifferenttransformants,andtheproductionofabell-curve

fortransgeneexpression.Oncethismethodcanbeusedtocreateareproduciblebell-

curveofluciferasedetection,thenstatisticalanalysiscanreliablydeducehowsmaller

changesareinfluencingproteinaccumulationlevels.

Figure 3.41 A comparison of means. By producing two bell curves with the luciferaseexpressionlevelmeasuredfrommanytransformants, itwillbepossibletoidentifysubtlerimprovementsinproteinaccumulation.Here!isthedifferencebetweentwotransformant

Num

bero

ftransform

ants

Luciferaseexpression

.

144

lines,withthebluelinerepresentingallthetransformantsofonetreatment,andtheredlineallthetransformantsofanother.

Development of the above will enable more precise testing of other strategies

including codon and codon pair optimization. The above also depends upon the

developmentofamoreefficientsystemoftransformingC.reinhardtiichloroplast,as

many transformantsneed tobe rapidlyproduced for thismethod tobeeffective.

Current work in the Purton lab by Saowalak Changko aims to improve current

transformationmethods.Anotherfutureadvantageofsuchasystemisthatitwould

allowtheidentificationofoutliers,cellswhichproduceveryhighorverylowlevelsof

recombinantprotein.Withthecostofwholegenomesequencing(WGS)fallingall

thetime,theseoutlierscouldbesubjectedtoWGSinanattempttounderstandwhat

factorsareinfluencingproteinexpressionsoheavily,andthenattempttospecifically

introducethosechanges.WGShasalreadybeenusedtosuccessfullyidentifypoint

mutationsinC.reinhardtii(Dutcheretal.2012).Thiswillimproveourunderstanding

ofthesubtletiesofcurrenttransformationmethodsandthecomplexroleofnuclear

factorsinchloroplastgeneexpression.

3.4.3 Codonpairoptimisation

As discussed in the previous section, attempting to manipulate and detect

differencesincodonpairoptimisationstrategiesmaybeprematureatthisstageof

developingtheC.reinhardtiichloroplastasaproteinproductionplatform.However,

giventheimportanceofcodonoptimizationoftenreportedintheliterature(Franklin

etal.2002;Surzyckietal.2009;Coragliottietal.2011),andtheclearexistenceofa

codonpairbiasintheC.reinhardtiichloroplast(Taunt2013),itispossiblethatcodon

pairoptimisationwillbecomeanimportanttoolinthefuture.

Codonandcodonpairoptimisationarecurrentlyjusta‘bestguess’athowtoexpress

arecombinantproteintoahighlevel.Itappearsthatcodonoptimisationbythehost

initsownendogenousgenesenablescontrolled,delicateadjustmentsofexpression.

Thediscoveryofcodonpairoptimisationfurthermuddiesthewatersaroundderiving

which codons to use in a transgene. It seems intuitive that just using the most

commoncodonswith the largestpoolof available tRNAswill result in the fastest

145

translationandthereforethegreatestlevelofproteinproduction.However,thereis

evidencetosuggestthatmoresubtlemechanismsareinplay,suchastheuseofrare

codonstoslowdowntranslationinsectionsoftheproteinthatrequirecomplexco-

translationalfoldingforexample(ShermanandQian2013).

Whenone is tryingtoexpressproteins forcommercialuses,generally thehighest

possiblelevelofproteinaccumulationisdesired,somethingthatthehostcellrarely

attemptstodoinnatura.Ontheotherhand,bacteriophage,whichinfecthostcells

and whose genomes follow similar codon usage patterns to their host, often do

attempttoproduceasmuchproteinaspossible,tomakeasmanyphageprogenies

aspossible(Lucksetal.2008).Inthecaseofendolysins,astheyaredesignedtobe

highlyexpressedinnatura,itcanbeassumedthattheyarealreadycodedinanear-

optimalmannerforthehostbacterium.Thisraisesthepossibilityofgeneratingan

inter-species codon map, by which one can map a rare codon in Streptococcus

pneumoniaetoararecodoninC.reinhardtiiforexample,thusconservingthepattern

of“fast”and“slow”codonsthathasbeenoptimisedovermillennia.

Overall, this study demonstrates that there is huge potential to improve protein

accumulation yields in the C. reinhardtii chloroplast, and the technique of using

multipleexpressioncassetteinsertionsappearstobearobustmethodofimproving

proteinyields.However,othermethodsofimprovingproteinaccumulationsuchas

codon pair optimisation firstly require improvements in understanding of clonal

variation and transformosome effects before they can be properly assayed and

implemented.

146

Chapter4 ImprovingtheactivityofCpl-1againstStreptococcus

pneumoniae

4.1 Introduction

ThischapteraimstoimprovetheactivityoftheStreptococcuspneumoniaetargeting

endolysin, Cpl-1, in its ability to lyseS. pneumoniae cells. Several strategieswere

testedwiththisaiminmind,namely:manipulatingthecellwallbindingsiteofCpl-1;

attemptingtoformpre-dimerisedCpl-1endolysinsintheC.reinhardtiichloroplast;

and investigating holin production in theC. reinhardtii chloroplast.While Cpl-1 is

alreadyahighlyactiveendolysin,withprovenefficacy invivousingmouseandrat

models(Loeffleretal.2003;Entenzaetal.2005;McCullersetal.2007;Grandgirard

etal.2008),anincreaseinactivitywouldenableareductionindose,thusminimising

some of the potential limitations discussed in 1.2.2.4 involving immunogenic

reactions.

4.1.1 ManipulatingCpl-1bindingactivity

Thefunctionofanendolysin,whenproducedbyabacteriophage,istolysethehost

bacterium, thus allowing the release of phage progeny into the environment in

search of another bacterial host. The endolysin, therefore, should lyse only the

current host cell, and not neighbouring bacterial cells, which could be potential

futurehosts for theprogeny.Onecharacteristicofendolysins is that theytendto

bindstrongly,ofteninthepico-tonanomolarrange(Loessneretal.2002),totheir

substrate - it has been postulated that this is a tethering mechanism to reduce

unintentionaldamagetonearbybacteria(Chapot-Chartier2014).

Thisfeature,whilebeneficialtobacteriophage,oftenreducestheactivitylevelofthe

endolysin as an enzyme as it restricts its lytic activity to a localised area of the

peptidoglycan cell wall (Nelson et al. 2001). For some endolysins, such as Cpl-1,

removal of the C-terminal cell wall binding domain (CBD) results in an almost

completelossoffunction(Sanzetal.1992),butforotherssuchasCD27L(aclostridial

endolysin)removingtheCBDcanimprovelyticactivityandbroadenthespectrumof

147

bacteria against which it is active (in this case, to include two more species, in

additiontothesixspeciessusceptibletothefull-lengthCD27Lprotein)(Mayeretal.

2011).

Whileitisclearfromtheliterature(Sanzetal.1992;Garcíaetal.1999;Monterroso

etal.2008)thatacompleteremovaloftheCpl-1CBDresultsinareductioninactivity,

we propose that if binding could be weakened, activity may be improved. With

weakerbinding,theendolysinmaybeabletoreleaseafterlysisandrepeatitslytic

actionselsewhere,thusmakingtheendolysinmoresuitedtoatherapeuticrole.This

project involved mutating the cell wall binding site of Cpl-1 via site-directed

mutagenesis,andcomparingtheactivityof theresultingproteinswiththatof the

original.

Itisworthnotingthatinthiscaseofmanipulatingthecellwallbindingsite,weare

usingaslightlydifferentdefinitionof“activity”.TheSIunitforenzymeactivityisthe

katal,1katal=1mols-1,i.e.thenumberofmolesofsubstrateconvertedpersecond.

However,inthisinstance,wearemoreinterestedinthenumberofmolesconverted

perendolysinmolecule,whichiswhatistermed“activity”inthischapter.

From a protein engineering perspective, Gram-positive-targeting endolysins are

particularly attractive due to their modular structure, and the decoupling of the

bindingandcatalyticdomains.Inmanyglobularenzymes,thebindingandcatalytic

sitesarethesame,suchthatchangestobindingsitesoftenreducetheabilityofthe

enzymetocatalysethereaction.Whiletheendolysincatalyticdomainmuststillbind

its substrate, the majority of binding specificity and affinity is conferred by the

bindingdomain(Mayeretal.2011).

4.1.2 Dimerisationasamethodtoimproveproteinstability

Cpl-1exhibitsextensivesimilaritieswithbothmajorpneumococcalautolysins,LytA

andLytC.Inthepresenceofcholine,LytAdimerises–becomingstableandactive,

whileLytCisactiveasamonomerbutisstabilizedinthepresenceofcholine.Cpl-1is

believedtodimerisethroughitsC-terminal(cellwallbinding)domainwhencholine

ispresent.Theadditionalactivityofthedimersislikelytobeduetothedoublingof

148

FigureRemoved

activesitesspatiallyorientatednexttothepneumococcalcellwall(Monterrosoetal.

2008).

Figure4.1ThestableandactiveLytAdimer.Cellbindingdomainsareshowninorange,thecatalyticdomaininblueandthe10cholineligandsshowninCPKcolourscheme.Reschetal.(2011)postulatethatCpl-1coulddimerise inasimilarmannergiventhehomologyof thefinal13aminoacidsattheC-terminus.ImagereproducedfromMaestro&Sanz(2016).

Cpl-1 is known tobe cleared from thebloodplasmaofmice in approximately20

minutes(Witzenrathetal.2009),presentingapossibleissueforsystemicuse.Resch

etal.(2011)hypothesisedthata“pre-dimerised”Cpl-1homodimerwouldbeamore

stable and active therapeutic than thenativemonomer. LytAdimerisationoccurs

throughthe13aminoacidsattheC-terminus(Fernández-Torneroetal.2002),and

10ofthese13aminoacidswerefoundtobeidenticaltotheC-terminusofCpl-1.By

themutationofeachofthe13aminoacidsinturn,Reschetal.(2011)produceda

seriesofmutatedCpl-1proteins,inanattempttofindonewhichformsadisulphide

bridgebetweentwomonomerstoproduceahomodimer.Thesuccessfulmutated

Cpl-1dimerwasshowntohavea10-foldreductioninplasmaclearanceandatwo-

foldincreaseinactivity,comparedtothemonomericversion(Reschetal.2011a).

Cpl-1naturallydimerisesonbindingcholine,thecrystalstructureofwhichhasbeen

reportedbyBueyetal.(2007)andisdisplayedinFigure4.2.Thisdimerisationoccurs

notattheC-terminus,asthepre-dimerisingmutantcreatedbyReschetal.(2011)

149

FigureRemoved

does,butbetweenthefinalcholinebindingrepeatsattheN-terminusofthecellwall

binding domain – i.e. the choline binding repeat closest to the acidic linker and

catalyticdomain.

Figure4.2TheC-terminusoftheCBDiscircledinredineachmonomer.Theboxshowstheside-chainof thearomatic residue involved indimerisation.Reproduced fromBueyet al.(2007).

Figure4.2doesnotshowtheacidiclinkerjoiningthetwodomainsofeachmonomer

as this could not be accuratelymodelled. This observedmode of dimerisation is

similartothatofthedimer-dimerinteractionsofLytAtoformatetramer(Bueyetal.

2007).

ThequaternarystructureofLytAandLytCiscrucialtotheirhydrolyticactivity,and

given the homology each displays to Cpl-1, it is reasonable to believe that the

quaternarystructureofCpl-1isalsoimportant.However,giventhestabilityofCpl-1

150

bothasamonomerandwithoutthepresenceofcholine,itmaynotbeasimportant

aswiththepneumococcalautolysins.

4.1.3 Theroleofholinsinthebacteriophagelyticcycle

Inthecanonicaltwo-genemodelofbacteriophage-mediatedcelllysis,theholinplays

anessentialroleinenablingtheendolysintoaccessthepeptidoglycancellwall,by

creatingahole in thecytoplasmicmembraneof thebacterium(Young1992).The

timingof this event is critically important to the success of thebacteriophageby

controlling the length of the cycle, and thus the holin gene is under enormous

selectivepressure(Wangetal.2000).Itisperhapsduetothisselectivepressurethat

holins are such a diverse functional group, comprising seven superfamilies

encompassing52recognisedfamilieswithnoclearconservedsequencemotif(Reddy

and Saier 2013). However, all holins have certain characteristics including the

possessionofatleastonetransmembraneα-helicalsequenceandahighlycharged

hydrophilicC-terminaldomain(Guetal.2014).

The release of endolysins beyond the cytoplasmic membrane is a carefully

orchestratedevent,andonewhichisnotwhollyunderstood.Severaltheoriesexist

onhowthistimingiscontrolled,butthemostwell-establishedtheoryisthatthereis

alateexpressionofholins,whichaccumulateinthecytoplasmicmembrane.When

theconcentrationoftheseholinsreachesacriticalthreshold,araft isformedand

thenaconformationalchangecausestheformationofahole(Whiteetal.2011).This

sequenceofeventsisillustratedinFigure4.3.

151

FigureRemoved

Figure 4.3 An illustration of the holin triggering mechanism, and the consequentialdepolarisationofthemembrane.pmf=protonmotiveforce.

Afurthermethodofholincontrolexistsinsomebacteriophagesystems,involvingan

antiholin.Anantiholinisalmostidenticaltotheholin,butwithoneortwoadditional

N-terminalaminoacids,oneofwhichisapositivelychargedresidue(usuallylysine)

andistranslatedfromadualstartmotifencoding:Met-Lys-(x)-Met-….Theantiholin

thenbindstotheholinresultinginheterodimersunabletoformtheraftdisplayedin

Figure4.3.However, thisbinding isPMF (protonmotive force)-dependent:as the

antiholin isproducedataslightly lowerrate thantheholin,aholewilleventually

form,resultinginaloweringofthePMFandtheunbindingoftheanti-holinanda

positive-feedbackresultinginthecollapseofthePMFandthe‘allornothing’release

ofendolysinsbeyondthecell(Pangetal.2013;Lellaetal.2016).

Thegenecph1,encodingaholin,islocatedimmediatelyupstreamofcpl1intheCp-

1 bacteriophage genome. The two genes are transcribed from two tandem

promotersupstreamofcph1andareexpressedfromlatephagetranscripts(Martín

etal.1998).Thecph1genedoesnotdisplaytheclassicaldual-startmotif,althoughit

doesencodeanothermethionineresidueatpositionsix,butno lysineresidue lies

betweenthem.Cph-1belongstotheHolinIVsuperfamilyandhasthreepredicted

transmembraneα-helicalsegments(ReddyandSaier2013).

Recent literature has suggested that holins can be applied exogenously to

Streptococcussuis(Shietal.2012)andStaphylococcusaureus(Songetal.2016)in

152

conjunctionwithendolysinstoproduceanenhancedantibacterialeffect,improving

thekillingefficacyandalsobroadeningthespectrumofsusceptiblepathogens(Song

etal.2016).ThestudiesbyShietal.(2012)demonstratethatthetransmembrane

domainsoftheholinareessentialforitsantimicrobialactivity,anditisassumedthat

theformationoflargeholesinthemembranecausescelldeath.However,itisnot

understoodhowtheholingainsaccesstothecellmembranegiventhepresenceof

the peptidoglycan layer, nor how capsules and slime-layers affect holin access.

Furtherinvestigationofholinstructureanditspotentialasanovelantibacterialhas

been hindered by the difficulties involved in successfully expressing holins in

prokaryoticsystems,giventhecytotoxicityofholins(Garrettetal.1990).Therefore

weaimtoproduceholinsintheC.reinhardtiichloroplasttoenablefurtherresearch.


1. To identify and optimize a method for comparing activity levels between

differentmutantCpl-1endolysinsagainstS.pneumoniae.

2. ToproduceseventransgenicC.reinhardtiilinesexpressingcpl-1geneswith

differentmutationsintheircholinebindingsites(CBS).

3. ToconfirmaccumulationoftheCBSmutantCpl-1proteinsintheC.reinhardtii

chloroplastbywesternblotting.

4. To compare the activity of the CBS mutant Cpl-1 proteins against S.

pneumoniae.

5. To produce a transgenicC. reinhardtii line expressing a variant cpl-1 gene

encodingadimerisingmutant.

6. ToconfirmaccumulationofaCpl-1dimerintheC.reinhardtiichloroplastby

westernblotting.

7. To produce the holin Cph-1 in the C. reinhardtii chloroplast and confirm

accumulationbywesternblotting.

153


4.3.1 ManipulationoftheCpl-1cholinebindingsitestoimproveactivity

The two choline binding sites on the C-terminus of the Cpl-1 protein recruit the

endolysintothecellwallbybindingtotheintegral(lipo)teichoicacids.Herethekey

residuesinvolvedinCpl-1cholinebindingareidentifiedandmutatedbysitedirected

mutagenesis.TheresultingCpl-1mutantsareassayedforlyticactivity.

4.3.1.1.1 Identificationofpotentiallyimportantresiduesincholinebinding

ThebindingaffinityoftheCpl-1cellbindingdomaintocholineisrelativelyweakfor

anendolysin,Kd≈3.6mM (Monterrosoetal. 2008),however the requirementof

bacteriophagestotethertheirendolysinstoonlythehost’scellwallsuggeststhatit

is unlikely to completely dissociate, and Cpl-1 possibly transits across the

glycopeptidenetworktoreachnewcleavablebonds(Jervisetal.1997;Monterroso

etal.2008).Inordertotestwhetherthereisanoptimalbindingaffinitythatimproves

activity,andliesbetweenthetwoextremesofcompleteCBDremovalandthenative

tetheringCBD,agradedweakeningoftheCBDwastested.

ThecrystalstructureofCpl-1bindingcholine,showninFigure3.1,wasanalysedusing

Jmol1.Interactionsoflessthan5angstromsfromthecholinemoietywereconsidered

to be of importance for hydrogen bonding during endolysin binding and two

tryptophanresiduesandonetyrosinewereidentifiedineachbindingsitetofitthese

criteria. This confirmed the original analysis by Hermoso et al. (2003)which also

identifiedthesethreeresiduesasimportant,formingahydrophobiccavityintowhich

thecholinemethylgroupsits.Hydrogenbondsformbetweenthethreeresiduesand

thecholineandtheseinteractionsareshowninFigure4.4.Forthepurposesofthis

investigation, the two tryptophan residues in each binding sitewere selected for

mutation.

1 Jmol is an open-source Java viewer for chemical structures in 3D.http://www.jmol.org/

154

ThecellwallbindingdomainofCpl-1hassixcholinebindingrepeats(p1-6,seeFigure

4.5),at the interfaceofwhichaputativecholinebindingsiteexists.However, the

crystalstructureofCpl-1suggeststhatonlythefirsttwocholinebindingsitesarein

factactive,namely:p1/p2andp2/p3(Hermosoetal.2003).Atthep1/p2site,there

aretwokeyinteractions:thenitrogenatomsonTrp202andTrp209interactingwith

thecholinemoiety.Atthesecondcholinebindingsite,thep2/p3site,itisTrp223and

Trp230thatinteractwiththecholine.WhenthefactthatanO-HbondandN-Hbond

are approximately one Angstrom in length is accounted for, the hydrogen bonds

formed in these interactions cover approximately 3-4 Angstroms. According to

Jeffrey (1997), hydrogen bonds over this distance range from “moderate,mostly

electrostatic”to“weak,electrostatic”.

Figure 4.4 The two active choline binding sites in the CBD of Cpl-1. Distances are inAngstroms.Itshouldbenotedthatthedistancesarebetweenthenitrogen(blue)andoxygen(red)atoms,soarenotadirectrepresentationofahydrogenbondlengthasthehydrogenmust be accounted for too in each case. However, these remain “moderate/weak andelectrostatic”hydrogenbonds.

Given that tryptophan is a polar amino acid, and it is this polaritywhich creates

hydrogenbonds,itwasdecidedtomutatethesetotheaminoacidalanine,whichis

non-polar.Thismethodofsystematicallysubstitutingresiduesforalanineisknown

as ‘alaninescanning’andisawidelyusedmutagenesisapproach(Kristensenetal.

p2/p3bindingsitep1/p2bindingsite

155

FigureRemoved

1997).Thedisruptionofthesehydrogenbondswasdesignedtoweakentheaffinity

ofCpl-1tocholine,andpotentiallyimprovethelyticactivityoftheendolysin.

4.3.1.2 ProductionofC.reinhardtiilinesexpressingcpl1CBSmutants

4.3.1.2.1 Site-directedmutagenesisofcpl1

Ofthefouridentifiedtryptophanresidues,shownaspartofthewholeCpl-1amino

acidsequenceinFigure4.6,noneofferedamoreorlessobvioustargetformutation

and therefore sevenC. reinhardtii cell lines expressing cpl1with either individual

tryptophanmutationsorcombinationsoftryptophanmutationsweregenerated.

Figure 4.5 The six putative choline binding repeats of Cpl-1. Asterisks indicate strictlyconservedresidues,whileacolonindicatesconservativesubstitutions.Redcircleshighlightthemutatedresidues.ReproducedandannotatedfromMonterrosoetal.(2008).

156

Figure4.6identifiesthefourmutatedtryptophanresiduesinthecpl1gene.Theseven

genemutantswhichwereintendedtobecreatedaredisplayedinFigure4.7,with

each tryptophan codon (TGG) beingmutated by site directedmutagenesis to an

alaninecodon(GCT).

Figure4.6Thefourtryptophanresiduesidentifiedtobeofimportanceincholinebindingarehighlightedinyellowinthecontextoftheentireproteinsequence.W202andW209formpartofthep1/p2cholinebindingsite,andW223andW230formpartofthep2/p3site.TheHAtagusedforwesternblotdetectionishighlightedinpurple.

MVKKNDLFVDVSSHNGYDITGILEQMGTTNTIIKISEST

TYLNPCLSAQVEQSNPIGFYHFARFGGDVAEAEREAQFF

LDNVPMQVKYLVLDYEDDPSGDAQANTNACLRFMQMIAD

AGYKPIYYSYKPFTHDNVDYQQILAQFPNSLWIAGYGLN

DGTANFEYFPSMDGIRWWQYSSNPFDKNIVLLDDEEDDK

PKTAGTWKQDSKGWWFRRNNGSFPYNKWEKIGGVWYYFD

SKGYCLTSEWLKDNEKWYYLKDNGAMATGWVLVGSEWYY

MDDSGAMVTGWVKYKNNWYYMTNERGNMVSNEFIKSGKG

WYFMNTNGELADNPSFTKEPDGLITVAYPYDVPDYA

202 209 223 230

157

Figure4.7Thegenecpl1wassubjectedtositedirectedmutagenesistocreatesevenuniquemutants. The naming of each construct indicates themutation location and nature, e.g.cpl1_W202A-HAisthecpl1genewithtryptophan-202mutatedtoanalanineresidue,andtaggedwithanHAtag.Multiplemutationsareseparatedbyanunderscore.

A pair of primers was designed for eachmutation, as displayed below, with the

mutationsiteinlowercase,andstartandstopcodonsunderlined.cpl1.Fandcpl1.R

aretheprimersatthestartandendofthegene.



W202A_SDM.F: GTACAgctAAACAAGATTCAAAAG

W202A_SDM.R: CTTTTGAATCTTGTTTagcTGTAC

W209A_SDM.F: CAAAAGGTgctTGGTTTC

W209A_SDM.R: GAAACCAagcACCTTTTG

W223A_SDM.F: CCATATAATAAAgctGAAAAAATTG

Trp202 Trp209 Trp223 Trp230

TGG TGG TGG TGG

GCT TGG TGG TGG

TGG GCT TGG TGG

TGG TGG GCT TGG

TGG TGG TGG GCT

GCT GCT TGG TGG

TGG TGG GCT GCT

GCT GCT GCT GCT

cpl1_W202A-HA

cpl1_W209A-HA

cpl1_W223A-HA

cpl1_W230A-HA

cpl1_W202A_W209A-HA

cpl1_W223A_W230A-HA

cpl1_W202A_W209A_-W223A_W230A-HA

cpl1-HA

158

W223A_SDM.R: CAATTTTTTCagcTTTATTATATGG

W230A_SDM.F: GTGGTGTTgctTATTATTTCG

W230A_SDM.R: CGAAATAATAagcAACACCAC

Thefirstfourmutantgenes,whichhaveonlyoneW→Achange,wereproducedusing

site directed mutagenesis (SDM) as shown in Figure 4.8. These mutated DNA

sequenceswerethenusedastemplateDNAinsubsequentroundsofSMDPCRto

producethefinalthreegenes,whichhavemorethanonemutatedcodon.

Figure 4.8 Site directedmutagenesiswas performed as above tomutate the tryptophanencodingcodon,TGG,tothealanineencodingone,GCT.PrimersareshowninblueandPCRconditionsaredescribedin2.3.7.

Of thesevenmutants, sixweresuccessfully created.The transformationofE. coli

withcpl1_W223A-HAinanintermediatevector,pJet,wasrepeatedlyunsuccessful.It

isnotclearwhythisparticularmutantwassoproblematic,anditisdiscussedfurther

in4.4.1,butduetotimeconstraintsitwasnotpossibletoinvestigatethisfurther.

TGGACC

GCT

CGA

GCTCGA

GCTCGA

1st PCR

2nd PCR

Finalproduct

Cpl-1

159


TheresultingPCRproductsweredigestedwithSapIandSphIand ligated in-frame

intothepSRSapIvector.TheinsertionwasconfirmedbyDNAsequencing.


TheC. reinhardtii recipient lineTN72was transformedby theglassbeadmethod,

using the pSRSapI_cpl1 plasmids (2.3.13). The resulting transformed lines are

representedinFigure4.9.PhotosystemIIfunctionalityisrestoredbypsbHandthe

resulting transformants were capable of growing phototrophically on minimal

medium.Eachofthesixtransformedlineswasshowntoreachahomoplasmicstate

by PCR as described in 2.3.17. As a final step, the integrity of the cassette was

confirmedbyDNAsequencingofthePCR-amplifiedplastomeregion,asin2.3.18.

Figure 4.9 The resulting C. reinhardtii lines express the mutated cpl1 gene under theendogenous psaA exon 1 5’UTR/promoter. The successfully transformed colonies areselectedbytheirabilitytogrowphototrophicallyonminimalmedium.


WholecellextractfromeachofthesixC.reinhardtiicelllineswasseparatedbySDS-

PAGE and blotted to nitrocellulose membrane for western blot analysis. The

membrane was probed with anti-HA primary antibodies followed by IRDye®

secondary antibody to enabledetectionusing the LiCorOdyssey®CLx system.An

imageofthewesternblotanalysisisshowninFigure4.10andrelativequantification

ofCpl-1accumulationisshowninFigure4.11.TheerrorbarsinFigure4.11represent

thestandarddeviationinsignaldetectionwhenthisblotwasrepeated.

trnE2 psbH

5’psaA 1 3’rbcL

TN72_SR_cpl1_WxxxA-HAtransformant

MluI

cpl1_WxxxA-HA

160

Figure4.10WesternblotanalysisofeachofthesixC.reinhardtii linesexpressingmutatedcpl1. Visible levels of accumulation are seen in all cases. TN72_SR_cpl1-HA andTN72_SR_Controlareincludedaspositiveandnegativecontrols,respectively.

Figure4.11Quantificationoftwowesternblotanalyses.Ingeneral,thefirstfourcell linesproduce levels of protein accumulation comparable to the unmodified version, whileTN72_SR_cpl1_W223A_W230A-HA and TN72_SR_cpl1_W202A_W209A_W223A_W230A-HAaccumulatetolowerlevels,approximately30%ofthatobservedfortheothers.

TN72

_SR_

cpl1_W

202A

-HA

TN72

_SR_

cpl1_W

209A

-HA

TN72

_SR_

cpl1_W

230A

-HA

TN72

_SR_

cpl1_W

202A

_W20

9A-HA

TN72

_SR_cpl1_W

223A

_W23

0A-HA

TN72

_SR_cpl1_W

202A

_W20

9A_-22

3A_W

230A

-HA

TN72

_SR_cpl1-HA

TN72

_SR_Co

ntrol

Cpl-1(39kDa)

0

0.2

0.4

0.6

0.8

1

1.2

TN72

_SR_cpl1_W20

2A-HA

TN72

_SR_cpl1_W20

9A-HA

TN72

_SR_cpl1_W23

0A-HA

TN72

_SR_cpl1_W20

2A_

W20

9A-HA

TN72

_SR_cpl1_W22

3A_

W23

0A-HA

TN72

_SR_cpl1_W20

2A_

W20

9A_-W22

3A_W

230A

-HA

TN72

_SR_cpl1-HA

TN72

_SR_Co

ntrol

RelativeFluorescence

161

As discussed at length in 3.3.2.2.4.3, the reason for the difference in protein

accumulation levels isdisputable.However, for thepurposesof this investigation,

onlytherelativeaccumulationlevelsarenecessarytoensurethatequalquantitiesof

proteinwereusedinfutureassays.

4.3.1.3 ActivityofCpl-1CBSmutantsagainstS.pneumoniae

ThesixC.reinhardtiilinesdescribedabovewerecreatedwiththeintentionofaltering

Cpl-1 activity. To measure this activity several approaches were taken, namely:

turbidity reduction assays and colony forming unit assays, using both crude cell

extracts, and extracts enriched and concentrated by ammonium sulphate

precipitationorepitopetagenrichment.

4.3.1.3.1 Activityofcrudeextracts–turbidityreductionassay

OneoftheproposedadvantagesofusingC.reinhardtiiasarecombinantendolysin

productionplatformisthat,duetoitsGRASstatus,simplecrudeextractscouldbe

used therapeutically, particularly for topical applications, thus removing the

expensiverequirementtopurifythefinalprotein.

Itwas therefore investigatedwhethercrudeextractsof thesixCpl-1CBSmutants

described in this section display different levels of lytic activity towards S.

pneumoniae,whencomparedtotheactivityoftheun-mutatedCpl-1protein.

Crudeextractswerepreparedasdescribedin2.4.1.Inbrief,thesixC.reinhardtiicell

lines were grown simultaneously and their OD740 measured after 72 hours. The

cultureswerecentrifugedandthepelletresuspendedin20mMNaPibuffertogive

suspensionsofequivalentcelldensitymeasuredatOD740.Thecellswere lysedby

freeze-thaw and ultracentrifuged to remove the cell debris. The supernatantwas

stored at 4 °C and thiswas considered the “crude extract”. Fresh S. pneumoniae

cultures were prepared as described in 2.7.1.1 and resuspended in 20mMNaPi

buffertoanOD595=~0.8.

Ina96-wellplate,S.pneumoniaecellsandC.reinhardtiicrudeextractwerecombined

andthechangeinOD595measuredwithaplatereaderoveraperiodof150minutes

at37°C.200μlofresuspendedS.pneumoniaecellswereincubatedwith~50μlof

162

crudeextract.TheprecisevolumewasadjustedaccordingtodatashowninFigure

4.11,toensureanequalquantityofCpl-1proteinwasincludedineachwell.Crude

TN72_SR_Controlwasusedasanegativecontrol.

163

Figure4.12TurbidityreductionassayofS.pneumoniaewhenchallengedwithcrudeC.reinhardtiiextractscontainingdifferentCpl-1CBSmutants.ChangeinOD595isshownratherthanmeasuredOD595duetothedifferentvolumesofcrudeextractineachwell.Eachcombinationwasperformedintriplicateandtheaverageisshown.

-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

00 15 30 45 60 75 90 105 120 135

ChangeinOD(595nm)

Time(minutes)

TN72_SR_cpl1_W202A-HA TN72_SR_cpl1_W209A-HATN72_SR_cpl1_W230A-HA TN72_SR_cpl1_W202A_W209A-HATN72_SR_cpl1_W223A_W230A-HA TN72_SR_cpl1_W202A_W209A_W223A_W230A-HATN72_SR_Control TN72_SR_cpl1-HANaPiBuffer(20mM)

164

From Figure 4.12, it appears that the un-modified TN72_SR_cpl1-HA is themost

active endolysin, while all the mutated endolysins showed activity intermediate

between this and the negative control, TN72_SR_Control. The high level of lysis

observedbythebufferaloneissurprising.However,ithasbeennotedinprevious

endolysinactivityassaysusingcrudeextractsthatan increase inosmoticpressure

duetothepresenceoflargequantitiesofproteininthesuspensioncanleadtoan

inhibitionofcelllysis(Mayeretal.2010)-thisisexemplifiedherebythehighlevelof

autolysisobservedwhenNaPibuffer(20mM)isadded,comparedtothatseeninthe

negativecontrol.

FirstinspectionofthedatashowninFigure4.12suggeststhatalteringtheCBDofCpl-

1hasadetrimentaleffectuponenzymaticactivity.Whilethisresult isnotentirely

unexpected, given the issue of osmotic pressure affecting bacterial cell lysis,

combinedwiththedifferentlevelsofproteinaccumulationobservedinFigure4.11,

itwasdecidedthatturbidityreductionassayswithcrudeextractsareinsufficiently

precisetoenableacomparisonofthevariousCpl-1CBSmutantactivities.

4.3.1.3.2 EnrichmentofCBSmutantCpl-1byammoniumsulphateprecipitation

Inordertoaddresstheaboveconcerns,ammoniumsulphateprecipitationwasused

to partially purify and concentrate the Cpl-1 CBSmutants, providing an enriched

crude extract. The enriched extracts were separated by SDS-PAGE, blotted to

nitrocellulose membrane and probed with antibodies for western analysis. The

resultinganalysisandrelativequantificationaredisplayedinFigure4.13.

165

Figure4.13ThewesternblotanalysisofsixCpl-1CBSmutantsconcentratedandenrichedbyammoniumsulphateprecipitation,alongsidepositiveandnegativecontrols(TN72_SR_cpl1-HAandTN72_SR_Control,respectively).ImageStudioLite5.0softwareandLiCorOdyssey®CLxsystemenabledthequantificationofeachband,shownbeloweachlaneasabarchart.

4.3.1.3.3 Activityofextractsenrichedbyammoniumsulphateprecipitation-Colony

formingunitassay

The turbidity reduction assay shown in Figure 4.12 required fresh S. pneumoniae

culturestobegrownonthemorningofeachassay,whichoftenleadstodifficulties

inthereproducibilityoftheassay.Ontheotherhand,colonyformingunitassays,as

described in2.7.1.1,wereperformedusingsnap-frozenaliquotsofS.pneumoniae

suspensions,thusenablingamuchmorereproducibleandrobustassay.Thenumber

of colony forming units was counted by eye and when an uncountable lawn of

indistinguishablecolonieswaspresent,thiswascountedas“100”.Serialdilutionsof

TN72

_SR_

cpl1_W

202A

-HA

TN72

_SR_

cpl1_W

209A

-HA

TN72

_SR_

cpl1_W

230A

-HA

TN72

_SR_

cpl1_W

202A

_W20

9A-HA

TN72

_SR_cpl1_W

223A

_W23

0A-HA

TN72

_SR_cpl1_W

202A

_W20

9A_-22

3A_W

230A

-HA

TN72

_SR_cpl1-HA

TN72

_SR_Co

ntrol

0

20000

40000

60000

80000

100000

120000

140000

TN72

_SR_cpl1_W20

2A-HA

TN72

_SR_cpl1_W20

9A-HA

TN72

_SR_cpl1_W23

0A-HA

TN72

_SR_cpl1_W20

2A_

W20

9A-HA

TN72

_SR_cpl1_W22

3A_

W23

0A-HA

TN72

_SR_cpl1_W20

2A_

W20

9A_W

223A

_W23

0A-HA

TN72

_SR_cpl1-HA

TN72

_SR_Co

ntrol

Relativefluorescence

166

bacterial suspension containing endolysin were performed after one hour of

incubationat37°C,andthree10μldropsofeachdilutionwerespottedontoblood

agarplates.Themeancountacrossthethreespotswascalculated.Thedatafrom

Figure 4.13 enabled similar quantities of HA-tagged protein to be added to the

bacterialsuspension.

167

Figure4.14ColonyformingunitassayofS.pneumoniaecellstreatedwithCpl-1CBSmutantammoniumsulphate-enrichedcrudeextracts.

0

10

20

30

40

50

60

70

80

90

100

10^2 10^3 10^4 10^5 10^6 10^7

Colonyfo

rmingunits

DilutionFactor

TN72_SR_cpl1_W202A-HA



TN72_SR_cpl1_W202A_W209A-HA

TN72_SR_cpl1_W223A_W230A-HA

TN72_SR_cpl1_W202A_W209A_W223A_W230A-HATN72_SR_Control

TN72_SR_cpl1-HA

20mMNaPiBuffer

168

ThecolonyformingunitassayinFigure4.14confirmsthatTN72_SR_cpl1-HAismore

activethananyoftheCpl-1CBSmutants,howevertherestillexiststheproblemof

varyingosmoticpressureduetothevaryingconcentrationsofproteinpresent.While

equal quantitiesof endolysinhavebeenachieved, this hasprobably resulted in a

different levelof totalprotein,becauseammoniumsulphateprecipitationenables

the enrichment of all proteins within a range of solubility, which undoubtedly

includesmanyotherproteins.InordertoincludesimilarquantitiesoftheHA-tagged

Cpl-1CBSmutant protein, differing volumesof the ammonium sulphate enriched

extractwereused, thusaltering theosmoticpressureonthebacterialcells in the

suspension.Inthiscasetoo,itisworthnotingthatthe20mMNaPibufferiscausing

lysis to a greater extent than all of the extracts except for the unmodified

TN72_SR_cpl1-HA.

4.3.1.3.4 Proteinenrichmentviaepitopetags–HAtags

AllCpl-1CBSmutantsweretaggedwithanHAepitopetagfordetectioninwestern

blotanalysis,butitisalsopossibletousethisepitopetopurifytheprotein.Eachof

thesixCpl-1CBSmutantswaspurifiedusingananti-HAresin,andthefractionswere

spottedontoanitrocellulosemembraneforadotblot,asdescribedin2.4.3.After

probing with anti-HA antibodies and IR secondary antibody, the membrane was

analysedusingtheLiCorOdyssey®CLxsystem,showninFigure4.15

169

Figure4.15DotblotanalysisofHA-resinpurificationfractions.TheC.reinhardtiilineswereabbreviatedtojustthemutationforclaritye.g.‘TN72_SR_cpl1_W202A-HA’isjustlabelled‘W202A’. Mixed results were achieved, with W202A, W202A_W209A,W202A_W209A_W223A_W230A,andtheun-mutatedCpl-1beingtheonlystrainswheretheelutionhadvisiblequantitiesofHA-taggedprotein.

ItisnoteworthythatTN72_SR_Controlgivesastrongsignalforthecrudeextractand

flow-through,which suggests that there are background proteins fluorescing and

disguising the HA-tagged proteins in these fractions. However, these background

proteins are not present in the elution fractions. To confirm that the fluorescing

proteinisindeedtheendolysin,themostpromisingexamplesfromthedotblotwere

separatedbySDS-PAGE.Westernblotanalysisallowsrelativequantificationofthe

yieldachievedwiththispurificationmethod,showninFigure4.16.

W202A

W209A

W230A

W202A_W209A

W223A_W230A

W202A_W209A_W223A_W230A

TN72_SR_cpl1-HA

TN72_SR_Control

Crud

eExtract

Flow

through

Wash1

Wash2

Elution1

Elution2

Elution3

170

Figure4.16WesternblotanalysisofbothcrudeC. reinhardtiiextractsandthenthesameextract enriched using the HA tag. Only mutants which have visible enriched bands areshown.ThebandswerequantifiedusedtheLiCorOdyssey®CLxsystemandtheresultsareshown in the bar chart. The percentage of recovered protein ranged from 33% for theunmodified Cpl-1 to only 6% recovery for the Cpl1-W202A_W209A_W223A_W230A-HAprotein.

Only three of the Cpl-1 CBS mutants achieved sufficient levels of protein

accumulationtoenableacolonyformingunitassaytobeperformed.

The colony forming unit assaywas performed using the same batch of frozen S.

pneumoniae cells as in Figure 4.14. TheC. reinhardtii crude extracts and theHA-

purifiedelutionsweredilutedsuchthattheycontainedequalconcentrationsofHA-

taggedproteinbeforebeingused.Themethodcanbefoundin2.7.1.1.Thenumber

of colony forming units was counted by eye and when an uncountable lawn of

indistinguishablecolonieswaspresent,thiswascountedas100.Serialdilutionsof

bacterialsuspensionandendolysinwereperformedafteronehourofincubationat

37°Candthree10μldropsofeachdilutionwerespottedontobloodagarplates.The

countwasthenaveragedacrossthethreespots.

(kDa)

171

Figure4.17ColonyformingunitassayofS.pneumoniaecellstreatedwithCpl-1CBSmutantcrudeextracts(dottedline)andtheHA-resinpurifiedelution(solidline).

Generally,thecrudeextractsarelessactivethanthepurifiedextracts–althoughthe

purified TN72_SR_Control appears to achieve the second highest level of

antibacterialactivity.Thebuffercontrolsitsnearlyinthemiddleofthecrudeextract

and purified extract activities, which lends weight to the argument that osmotic

pressureisreducingcelllysisinthecrudeextracts.Theun-mutatedCpl-1proteinhas

thegreatestantibacterialeffect,whiletheCpl-1CBSmutantsarenomoreeffective

than the purified TN72_SR_Control, which expresses no recombinant protein,

suggestingacompletelossofactivityinthemutantproteins.Giventhepoorresults

achievedusinganti-HApurificationresin,enablingustoonlytestthreeofthesixCBS

mutants,adifferentsystemisrequired.

172

4.3.1.3.5 Proteinenrichmentviaepitopetags–StrepIItags

TheHAepitopehasbeenwidelyused in thePurton group, aswell as other algal

research groups. The original expression of cpl1 in C. reinhardtii encoded an HA

epitope tag on its C-terminus (Taunt 2013), and this was continued into this

investigation.However,whileconvenientforwesternblotdetection,usingtheHA-

tag for protein purification is expensive compared to some other epitopes, as it

involvesaffinitypurificationusingananti-HAmonoclonalantibody.TheStrepIItag

waschosenasanalternativeas ithasbeenused inthealgal fieldtopurifynative

chloroplast proteins from C. reinhardtii (Derrien et al. 2012; Derrien 2013).

Furthermore, as the system is based on the affinity of biotin and streptavidin,

purificationcolumnsarerelativelycheap.3.3.2.2.3describestheproductionoftheC.

reinhardtii line TN72_SR_cpl1-StrepII, which is used again here to investigate the

StrepII-tagforproteinpurificationandenrichment.Polyhistidinetags(His-tags)are

themostcommonepitopetagusedforthispurposebutaStrepIItagwaschosenin

thisinstanceduetoitsprevioussuccessintheC.reinhardtiichloroplast,aswellas

thefactthatitcanbereadilyusedfordetectioninwesternblotting,whileantibodies

toHistagsaremoreproblematic.

4.3.1.3.6 EnrichmentofCpl1-StrepIIusingHiTrapStrepIIColumn

C.reinhardtiicellextractswerepreparedasdescribedin2.5.1,andultracentrifuged

at100,000gfor1hourtoremovecelldebris.Thisultracentrifuged(UC)extractwas

then enriched for StrepII-taggedproteins using the commercially availableHiTrap

StrepIIColumn(GELifesciences)andelutedwithdesthiobiotin,asdescribedin2.5.3.

173

Figure4.18SamplesfromextractpreparationandtheHiTrapStrepIIcolumnwerecollectedandseparatedbySDS-PAGE.WesternblotanalysisandrelativequantificationusingtheLiCorOdyssey® CLx system enabled the StrepII-tagged protein recovery to be estimated as apercentageoftheultracentrifuged(UC)extractpassedthroughthecolumn.

Approximately one-third of the total protein remains in the supernatant on

ultracentrifugation,butvirtually100%ofthiswasrecoveredusingtheHiTrapStrepII

column. Further optimisation of ultracentrifugation speeds and durations could

increase the proportion remaining in the supernatant further, but for this

investigationmaximisingtotalproteinyieldisnottheaim.

Asanegativecontrol,HiTrapStrepIIpurificationwasalsoperformedwithanextract

ofTN72_SR_Control.Thiscontrols foranyC.reinhardtiicellcomponentsthatmay

havenon-specificallyboundtothecolumnandbepresentintheelutedfraction.

174

4.3.1.3.7 ActivityofpurifiedCpl1-StrepII

AS.pneumoniaecolonyformingunitassaywasperformedasdescribedpreviously

(4.3.1.3.4),usingtheelutedfraction,describedhereasan“enrichedextract”,from

bothTN72_SR_cpl1-StrepIIandTN72_SR_Control.Bacterialsuspensionandextract

were combined in a 1:1 ratio and serial dilutions of bacterial suspension and

endolysinwereperformedafteronehourofincubationat37°C,whereuponthree

10μldropsofeachdilutionwerespottedontobloodagarplates.

Figure 4.19 Colony forming unit assay of S. pneumoniae cells treated with Cpl1-StrepIIenrichedextracts.“Control”referstoapurifiedextractfromTN72_SR_Control.Thenumberof colony forming units was counted by eye and when an uncountable lawn ofindistinguishable colonies was present, this was counted as 100. The count was thenaveragedacrossthethreespots.Threedilutionsoftheenrichedextractweretested(100%,75 % and 25 %), and the percentage indicates the initial concentration, not the finalconcentration(i.e.priortomixingwiththebacterialsuspension).

175

The colony forming unit assay in Figure 4.19 indicates a measurable difference

betweenthethreeCpl-1dilutions,andtheyareclearlymoreactivethantheextract

fromTN72_SR_Controlatthetwohigherconcentrations.Thesedatasuggestthatthis

method of enrichment removes both the mild antibacterial activity of the C.

reinhardtiicrudeextractandtheosmoticpressureeffectthatseemedtobemasking

resultsinprevioustrials.

Unfortunately,timelimitationspreventedtheapplicationofthismorerobustassay

totheinvestigationofbindingsitemutation.ByreproducingeachoftheC.reinhardtii

linesexpressingCBSmutants,thistimeencodingaStrepIIepitopeinsteadofanHA

epitope, itwouldbepossibletomoreaccuratelycomparetherelativeactivitiesof

the mutants. However, given the problems encountered above and the longer

transformationtimeofC.reinhardtiilines,inthisparticularcaseinvestigationscould

bemoreeasilycarriedoutusingrecombinantproteinsproducedinE.coli.E.coliisan

excellentplatformforrapidlyproducingrecombinantproteinstoahighlevelandin

this case would allow easy comparison of different mutants. The best of these

mutants,ifindeedcellwallbindingsitemutagenesiscanimproveendolysinactivity,

couldthenbeexpressedinC.reinhardtiiforthefinaltherapeuticdrugproduction.In

addition, proteins toxic to E. coli, such as the endolysin SPN9CC, which can be

expressedinC.reinhardtii(YoungandPurton2015)couldbeinvestigatedusingthis

method.

176

4.3.2 ImprovingCpl-1stabilitythroughdimerisation

TheC. reinhardtii chloroplast is known to produce and regulate the formation of

disulphidebonds(Kim1997),andresearchsuggeststhatyieldsofproteinscontaining

disulphidebondscanbeimprovedintheC.reinhardtiichloroplastbytheadditionof

selenocystaminetotheculturemedium(Ferreira-Camargoetal.2015).Furthermore,

Resch et al. (2011) showed that Cpl-1 can be modified to “pre-dimerise”, thus

decreasingplasmaclearanceandimprovingitsclinicalpotential.

Cpl-1 is stable as amonomer, but Cpl-1 dimerisation occurs naturally on choline

binding (Buey et al. 2007). We expect that the addition of a disulphide bridge

between two Cpl-1 monomers will reduce the rate of degradation in storage by

limitingbothproteaseaccessandthedenaturingoftheprotein,aswellasachieving

thedecreasedplasmaclearancereportedbyReschetal.(2011).Theaimisalsoto

show that the C. reinhardtii chloroplast is a suitable platform for producing

disulphide-bondedendolysinsastherapeutics.

4.3.2.1.1 StabilityofCpl-1undervaryingconditions

AninitialinvestigationintothestabilityofCpl-1instoragewasconducted.Anextract

fromtheC.reinhardtiilineTN72_SR_cpl1-HAwaspreparedasdescribedin2.5.1.The

cellswere either resuspended and lysed in 20mMNaPi buffer or 20mMNaPi +

ProteaseInhibitorbuffer,thenbothwereultracentrifugedforonehourat100,000g.

Eachsupernatantwasthensplitintotwotubesandstoredat25°Cor4°Cforseven

days.Thefourtubesweresampledat24-hourintervalsandthesamplesweresnap-

frozeninliquidnitrogenandstoredat-80°C.Attheendoftheseven-dayperiod,all

samples were thawed and separated by SDS-PAGE before being blotted to

nitrocellulosemembraneandanalysedbywesternblotting.Relativequantification

usingtheLiCorOdyssey®CLxsystemproducedthedatadisplayedinFigure4.20.

177

Figure4.20Cpl-1proteindegradationoveraseven-dayperiodwhenstoredat4°Cor25°Cwithandwithoutproteaseinhibitor.

Thedatadisplayed inFigure4.20appearsveryerraticas thiswasonlyperformed

once,butingeneral itappearsthattheproteinisbestconservedat4°Candwith

proteaseinhibitor,whichwastobeexpected.Thishasestablishedabenchmarkfor

the stability of monomeric Cpl-1 when stored under lab conditions. If Cpl-1

dimerisationcanbeachieved,thenthisstabilitymaybeimproved,thusenablingthe

addition of less protease inhibitor which would reduce costs and simplify the

therapeuticregulatoryprocedure.

4.3.2.2 ProductionofaC.reinhardtiilineexpressingcpl-1dimer

The pre-dimerised Cpl-1 protein produced by Resch et al. (2011) was achieved

through site-directedmutagenesis, and their experiments are briefly summarised

here: site-directed mutagenesis firstly changed a cysteine residue near the N-

terminus(C45)toaserine(seeFigure4.21).Thisfirststepremovedthepossibilityof

178

interactions with this solvent-accessible cysteine. Then an aspartic acid residue

(D324), inaposition13aminoacidsawayfromtheC-terminus,wasmutatedtoa

cysteine. The 13 final amino acids are highly conserved and, in the case of the

homologouslysinLytA,responsibleforLytAdimerisation,sowerenotmodified.

4.3.2.2.1 Site-directedmutagenesisofcpl1

Sitedirectedmutagenesiswasperformedasdescribedin2.3.7.Theprimersusedin

thisinstancearebelow,andmutationsitesareinlowercase:


cpl1_C45S.F ACATATTTAAATCCATcaTTATCAGCTC

cpl1_C45S.R GAGCTGATAAtgATGGATTTAAATATGT

cpl1_D324C.F GTGAATTAGCAtgtAATCCTTCATTT

cpl1_D324C.R AAATGAAGGATTacaTGCTAATTCAC


179

Figure4.21Thetwoaminoacidsselectedformutationarehighlighted inyellow.C45wasmutated to a serine to prevent unwanted interactions, as this cysteine is believed to besolvent-accessible. D324 was mutated to a cysteine to form a disulphide bridge in ahomodimer.TheHAtagishighlightedinpurple.


ThefinalPCRproduct,namedcpl1_dimer-HA,wasinsertedintothepSRSapIvector

usingSapIandSphIaspreviouslydescribed(4.3.1.2.2).CompetentDH5αE.coliwere

then transformedwith the resultant plasmid and DNA sequencing confirmed the

correctinsertionandthattheintendedmutationshadbeensuccessfullyintroduced.


The TN72 recipient line was transformed with the pSRSapI_cpl1_dimer-HA

transformation plasmid. Transformed C. reinhardtii lines, named

TN72_SR_cpl1_dimer-HAwerecheckedforhomoplasmicitybyPCR(AppendixI)and

finallyconfirmedbysequencing.

180


Whole cell extracts of TN72_SR_cpl1-HA and TN72_SR_Control (as controls) and

TN72_SR_cpl1_dimer-HAwereprepared,bothwithandwithoutβ-mercaptoethanol

(BME), and separated by SDS-PAGE. The BME reduces disulphide bonds so was

includedtotestifthedimerisedCpl-1couldbedetectedwhenBMEwasabsentbut

not when present – this would confirm disulphide presence. After blotting to

nitrocellulose membrane and blocking as described in 2.4.5, the membrane was

probed with anti-HA primary antibody, then incubated with IRDye® secondary

antibody. Fluorescence was observed using the LiCor Odyssey® CLx system. The

resultinganalysisisshowninFigure4.22.

Figure4.22Westernblot analysisof TN72_SR_cpl1_dimer-HA. The loadingmixtureeithercontainedβ-mercaptoethanolordidnot,indicatedby+/-BME.ThepredictedbandlocationforCpl-1_dimerisindicatedbutnobandswereobserved.

No spontaneously forming Cpl-1 dimerswere observed in thewhole cell extract.

However, monomeric Cpl-1 still accumulates to similar levels as seen in

TN72_SR_cpl1-HA.Thepresenceofthemonomersuggeststhatthereisnoissuewith

synthesis,butforsomereasondimerisationisnotoccurring,perhapsduetosteric

issuesinvolvingtheC-terminalHAtag,orsimplythat,attheconcentrationsobserved,

theequilibriumisshiftedtowardsmonomersanddimersarenotformed.

(kDa)

181

4.3.2.2.5 Additionofselenocystamine

The addition of the smallmolecule selenocystamine to the growthmediumofC.

reinhardtii was reported to catalyse disulphide bond formation in recombinant

proteins(Ferreira-Camargoetal.2015).

SelenocystaminewasaddedtoC. reinhardtii cultures toa finalconcentrationof2

μM,theoptimalconcentrationobservedbyFerreira-Camargoetal. (2015).Whole

cell extracts of TN72_SR_cpl1-HA and TN72_SR_Control (as controls) and

TN72_SR_cpl1_dimer-HA, all grownbothwithandwithout2μMselenocystamine

wereprepared,withoutβ-mercaptoethanol(BME),andseparatedbySDS-PAGE.The

westernblotanalysis,carriedoutaspreviously,isdisplayedinFigure4.23.

Figure 4.23 Western blot analysis of whole cell C. reinhardtii extracts, grown withselenocystamine (+ Sel.) and without. All were prepared in the absence of β-mercaptoethanolandthepredictedCpl-1dimerbandlocationindicated.

TheadditionofselenocystaminetothegrowthmediumofTN72_SR_cpl1_dimer-HA

failedtocatalysespontaneousCpl-1dimerisation,nordiditsignificantlyaffectlevels

ofmonomericCpl-1accumulation.

(kDa)

182

4.3.2.2.6 Additionofcholine

The investigation by Resch et al. (2011) used DEAE-Sepharose™ affinity

chromatographytopurifyCpl-1fromE.coliextracts.DEAEactsasacholineanalogue

andthereforethismethodexploitsthecholinebindingabilityofCpl-1.Cpl-1wasthen

elutedfromthecolumnusingabuffercontaining10%choline.However,nocrude

lysateanalysiswasperformedbyReschetal.(2011)andonlythepurifiedCpl-1was

shownonSDS-PAGE.WhileitismostlikelythatintheexperimentsbyReschetal.

(2011)Cpl-1dimerisationoccurredinvivointheE.coliperiplasm,itispossiblethat

thedimerisationoccurredinvitrowhentheadditionoffreecholinerecruitedCpl-1

monomerstogether,enablingspontaneousinvitrodimerisation.

Totestthishypothesis,freecholinewasaddedtocellextractcontainingCpl-1_dimer,

atvaryingconcentrations,andincubatedforonehour.Thesampleswereseparated

by non-reducing SDS-PAGE and analysed bywestern blotting, displayed in Figure

4.24.

Figure4.24WesternblotanalysisofwholecellC.reinhardtiiextracts,incubatedwithcholine.Thefirstbandhasnocholineadded.Allwerepreparedintheabsenceofβ-mercaptoethanolandthepredictedCpl-1dimerbandlocationindicated.

IncubationwithcholinefailedtoinduceCpl-1dimerisation.Thepresenceofcholine

wasexpectedtorecruitCpl-1towardsthefreecholineandenabletheformationof

adisulphidebondbetweenthetwomonomers.ThenativedimerisationofCpl-1upon

(kDa)

183

theadditionofcholinereportedbyMonterrosoetal.(2005)wasnotexpectedtobe

observed,giventhedenaturingconditionsofSDS-PAGE.

4.3.2.2.7 ProductionofCpl-1_dimerinE.coliandenrichment

As shown in Figure 3.8, the E. coli line DH5α_pProEX_cpl1_dimer-HA-StrepII was

shown to express Cpl-1 to a high level, and the application ofHiTrap StrepTactin

purification columns enabled the production of an enriched extract. This was

performedtoreproducetheworkofReschetal.(2011),aswellastoactasaprotein

standardasdescribedin3.3.1.2.6.WhenseparatedbySDS-PAGEundernon-reducing

conditions,nodimerisationcouldbeobserved,howeverthiswastobeexpectedas

disulphidebondformationrequiresanoxidisingenvironment inordertooxidisea

pairof cysteines. InGram-negativeprokaryotes, suchasE. coli, thisoccurs in the

periplasmic space, where enzymes catalyse the reaction. Recombinant proteins

thereforerequireanN-terminalsignalpeptidetotargetthemtotheperiplasmfor

disulphidebond formation,whichwasnot includedonourcpl1_dimer transgene.

Resch et al. (2011) expressed cpl1 using a plasmid based upon pIN-IIIA, which

attachesanoutermembraneproteinA(OmpA)signalpeptidetotheN-terminusof

clonedproteinstopromotesecretionoftherecombinantproteinintotheperiplasm.

Giventhattheplasmidusedhere,pProEX_HTb,doesnotattachanN-terminalsignal

peptide,itwasnecessarytoperformdimerisationinvitro.

4.3.2.2.8 DimerisationofE.coli-producedCpl-1usingoxidisedglutathioneinvitro

Disulphidebondformationcanoccurspontaneouslyiftheenvironmentisoxidising,

andcan thereforebe formed invitro (Berkmen2012).Glutathione isacommonly

used reagent in its reduced (GSH) and oxidised (GSSH) form to produce a redox

environmentbeneficialtodisulphidebondformation.Theprotocolisdescribedfully

in2.6.5,adaptedfromHidakaetal.(1998)andHyvonen(2002).

A crude cell lysateofE. coli cells induced toexpresscpl1_dimerwasobtainedby

sonication,andultracentrifugationremovedtheinsolublefraction.AnexistingCpl-

1_StrepIIenrichedextractfromC.reinhardtiiwasusedasacontrol(from4.3.1.3.6).

The soluble fractionand thecontrolwere then incubatedovernightat20 °Cwith

varyingGSH:GSSGratios.SDS-PAGEwasusedtoseparatethefractions,withnoβ-

184

mercaptoethanolandnoheatdenaturationprior torunningthegel.Theresulting

westernblotanalysisisshowninFigure4.25.

Figure4.25WesternblotanalysisattemptingtodetectinvitrodimerisationofCpl-1_dimer,withCpl-1_StrepIIbeingusedasanegativecontrol.TheCpl-1_dimerrunsslightlylargerthantheCpl-1_StrepIIduetotheadditionalepitopetagsandslightlydifferentcharge.Thesampleinthefirstlaneofeachblotwasboiledfor3minutesinthepresenceofβ-mercaptoethanol,whichshouldreduceanydisulphidebonds.Thesubsequent lanesallcontain2.5mMGHSandincreasingquantitiesofGSSG,from0mMto5mM.The‘?’markstheexpectedlocationofthe78kDaCpl-1dimerband,butasdiscussedbelow,theobservedbandisnotbelievedtobethedimer.

Despitethepresenceofa78kDaband,whichisthepredictedsizeofaCpl-1dimer

(Reschetal.2011a),thisisnotbelievedtobeaCpl-1dimer.Thepresenceofthe78

kDabandinthefirstlane,wherethesamplehasbeenboiledinthepresenceofβ-

mercaptoethanol,suggeststhatthisisanartefactofnon-specificbindingoftheanti-

StrepIIantibodyratherthandimerisedCpl-1.Itispossiblethatthisbandismasking

Cpl-1 dimer formation in subsequent lanes, however this seems unlikely, as one

wouldexpecttobeabletoseetwobandseveniftheywerelocatedclosetogether

or the single band would become brighter. No non-specific band is seen in the

negativecontrol,butthisisanextractfromC.reinhardtiithathasbeenpurifiedusing

aStrepTrapcolumnaspreviouslydescribed,sohasadifferentbackgroundprotein

make-uptotheE.coli-derivedcrudeextract.Itservesitspurposeofshowingthatno

dimerisationoccurswiththeun-mutatedCpl-1,whiletheβ-mercaptoethanollaneis

anegativecontrolforthemutatedCpl-1.

(kDa)

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ThereareacoupleoffactorsthatcouldbepreventingdimerisationofCpl-1_dimer,

eveninvitro.ThefirstandmostlikelyissueisthepresenceofC-terminalepitopetags

causingsterichindrance.TheHAtagintheC.reinhardtiiversionoftheproteinand

theHAandStrepIItagsintheE.coliversionarebothontheC-terminusoftheprotein.

Inhindsight,locatingtheseontheN-terminusmayhavebeenabetterapproachas

theCpl-1proteinhasbeenengineeredtoformadisulphidebridgeclosetotheC-

terminus.Reschetal.(2011)notethatthepositioningofthenewcysteineresidue

neartheC-terminusisofcrucialimportancetodimerisation.Givenallofthis,itisnot

surprisingthattheinclusionofan8-9aminoacidepitopetagattheC-terminuswould

interferewithdimerisation.

ThesameCpl-1_dimerproteinwasexpressedasarecombinant inE.coliafterthe

failure to achieve dimerisation inC. reinhardtii to testwhether this failurewas a

propertyoftheproteinperseoraresultoftheexpressionplatform.Inessence,if

dimerisationofCpl-1_dimercouldbeachievedinE.colibutnotinC.reinhardtiithen

wewouldconcludethattheC.reinhardtiichloroplastisunsuitableforpre-dimerised

Cpl-1production.However, ifdimerisationcouldnotbeachieved inE.colieither,

thentheconclusionwouldbethattheproteindesignwasinsomewaypreventing

dimerisation.

In this study, the lackof targeting to theE.coliperiplasm likelyprevented invivo

dimerisationfromoccurring.FuturestudiesshouldusevectorsuchaspIN-III,which

includesanN-terminalOmpAtargetpeptideandmayrectifythis,orconsiderusing

anengineeredstrainofcompetentE.colisuchasRosetta-gami™(Novagen)which

displaysenhanceddisulphidebondformationinthecytoplasm.Thefailureofinvitro

dimerisation could be due to a lack of condition-optimisation of the dimerisation

protocol,asonlyarelativelynarrowrangeofconditionsweretested.Forexample,

differentreagentscouldbeused,suchascysteineandcystine,andindifferentratios

tocreatetheredoxenvironment,aswellasdifferenttemperaturesandincubation

times.OptimisationofthisprotocolforCpl-1_dimermayhaveenableddimerisation.

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4.3.3 ExpressionoftheCpl-1associatedholin,Cph-1

4.3.3.1 ProductionofC.reinhardtiilinesexpressingcph-1

TheC.reinhardtiiderivedpsaA5’UTR/promoterisfunctionalinE.coli,asareseveral

chloroplastpromotersprobablyduetothechloroplast’sprokaryoticorigins(Young

and Purton 2015). Given the ability of holins to form holes in the cytoplasmic

membraneofE.coli,andthusinhibitcellgrowth(Catalãoetal.2011),theuseofthe

pSRSapIplasmidtoclonetheseinE.coliisunlikelytobepossible.

AstrategydevelopedbyYoung&Purton (2015)makesuseofa ‘spare’opal stop

codon,UGA,thatisunusedintheC.reinhardtiichloroplast.Thecodonisreassigned

tocodeforatryptophanbytheco-introductionofamodifiedtryptophantRNAgene

(trnW)intotheC.reinhardtiichloroplast,alongwiththegeneofinterest.Thegene

of interest is modified to contain several UGA codons in the place of UGG

(Tryptophan)–thesearereadas“Stop”codonsbytheE.colitranslationalmachinery,

but as tryptophan codons in the chloroplast. A plasmid, named pWUCA2 was

developedtoperformthiscloningstrategy.Theplasmid isverysimilar topSRSapI

(2.2)butwiththeadditionofatrnWUCAgeneupstreamofthegeneofinterest(GOI)

expressioncassette,illustratedinFigure4.26.

Figure4.26ThepWUCA2 transformationvector isbaseduponpSRSapIandhas thesamecloning sites for the GOI and the same flanking regions for homologous recombination.However, an additional synthetic gene, trnWUCA, has been inserted upstream of the GOIexpressioncassette.

This strategy enables the cloning inE. coli of genes under functional/constitutive

promoters,whichwouldotherwiseproduceproteinsthataretoxicordetrimentalto

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E. coli growth. In this case, the E. coli translational machinery will stop short of

producingthewholelengthproteinduetothepresenceofstopcodonsinthemiddle

oftheGOI.

ProducingholinsintheC.reinhardtiichloroplastmayavoidtheproblemofE.colilysis

thatoccurswhenholinsareexpressedasrecombinantproteinsinbacterialplatforms

(Smithetal.1998;Shietal.2012;Songetal.2016).Thisproblemhasbeenlargely

overcome by the use of induction systems, but allows only 10 – 15 minutes of

accumulationbeforethemetabolicactivityfails(Smithetal.1998).Furthermore,the

natureoftheproteinmeansthatitmustbepurifiedfromthebacterialmembraneto

which it localises, which further reduces yields and increases costs. While C.

reinhardtiiachievesonlylowlevelsofrecombinantproteinexpressioncomparedto

E.coliformanyproteins,giventhesetwoissuesitmaybethatitcancompeteasan

expressionplatformwhenproducingholins.

4.3.3.1.1 Genedesignandcodonoptimization

CodonUsageOptimizerBeta0.92(Kong2013)wasusedtooptimizethecph1gene

forC.reinhardtiichloroplastexpression.Twotryptophancodonsearly inthegene

werechangedfromUGGtoUGA.SapIandSphI restrictionsiteswere includedfor

cloning into the pWUCA expression vector. The full-length cph1-HA gene was

synthesizedbyIntegratedDNAtechnologies(AppendixH).


SapIandSphIrestrictionenzymeswereusedtodigestandligatethecph1geneinto

pWUCA2 in-frame,producing theplasmidpWUCA2_cph1-HA.DH5αcompetentE.

colicellsweretransformedwiththeresultingplasmidandsequencingconfirmedthe

correctplasmidassembly.


ThepWUCA2_cph1-HAplasmidwasused to transform the recipientC. reinhardtii

strainTN72viatheglassbeadmethodasdescribedin2.3.13.Figure4.27illustrates

theresultingC.reinhardtiicellline,pWUCA2_cph1-HA.

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Figure4.27TN72recipientstrainsuccessfullytransformedtocarrycph1-HAunderthepsaAexon1promoter/5’UTRinthechloroplast,andwithpsbHfunctionalityrestored.AmodifiedtrnWgenehasbeenco-introducedtocorrectlycodefortheintroducedUGAcodonsinthecph1gene.TheapproximatepositionsoftheUGAcodonsareindicatedinred.

The resulting transformants were confirmed for homoplasmicity as described in

2.3.17.Inthisinstance,duetotheadditionofthetrnWUCAgene,theexpectedband

sizeforhomoplasmicitywillbe291bplarger,givingatotalexpectedsizeof1421bp

(Primers:Flank1&psa.R (AppendixO:ListofPrimers)).Gelelectrophoresisof the

resultingPCRproductfromthreetransformantsisshowninFigure4.28.

Figure4.28TheresultingPCRproductsareapproximately1.4kbinsizeasexpected.Thelackofan850bpproductconfirmsthatallthreeofthesetransformantsarehomoplasmic.

Finally,theintegrityoftheinsertedcassettewasconfirmedbyDNAsequencingasin

2.3.18.


WholecellextractsofTN72_WUCA2_cph1-HAandTN72_SR_Control(asanegative

control)werepreparedandseparatedbySDS-PAGE.Afterblottingtonitrocellulose

membraneandblockingasdescribedin2.4.5,themembranewasprobedwithanti-

HAprimaryantibody,thenincubatedwithIRDye®secondaryantibody.Fluorescence

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wasobservedusingtheLiCorOdyssey®CLxsystem.Theresultingimageisshownin

Figure4.29.

Figure4.29Westernblotanalysis indicates thata~15kDaprotein isdetectedbyanti-HAantibodies in the TN72_WUCA2_cph1-HAextract, andnot in thenegative control. This islikelytobeCph-1protein.

Cph-1 appeared to be present in TN72_WUCA2_cph1-HA whole cell extract.

However,itwasnotedthatthetransformedC.reinhardtiilinesdidnotappeartobe

as healthy as other lines, in that they took a longer time to reach a bright green

culture,takingapproximatelysixdaysratherthantheusualthreeorfour.Thiswas

merely an observation, and no data was collected at the time to objectively

investigateachangeingrowthrate.

4.3.3.1.5 Investigationintoholinlocalisation

Giventhenon-specific insertionofholins intobacterialcellmembranes, it is likely

that the holin would insert into chloroplast membranes when produced as a

recombinant. In order to investigate whether the holin was integrating into the

thylakoidmembraneorchloroplastenvelope,TN72_WUCA2_cph1-HAwaslysedby

sustainedglassbeadagitationat4°Candsubjectedtoultracentrifugationat100,000

gfor1hourat4°C.Thiswasdesignedtoseparatethemembranefraction(pellet)

(kDa)

190

and cytoplasmic fraction (supernatant). However, when the two fractions were

separated by SDS-PAGE and analysed by western blotting as before, no 15 kDa

proteincouldbedetected.

InanattempttodetectCph-1accumulationagain,veryhighconcentrationsofcell

extractwereseparatedonSDS-PAGEandanalysedbywesternblotting,aswellas

usingammoniumsulphateprecipitationtoconcentratelargevolumesofcellextract,

butnoCph-1wasdetected.Again,onlyobservationally,itwasnotedthatatthetime

ofthesefurtherexperiments,theTN72_WUCA2_cph1-HAcelllineseemedtogrow

atanormal rateand ithadrecovered from itsearlierunhealthyappearance.This

observationandthelackofdetectableCph-1accumulationsuggestedthatthegene

hadbeensilenced insomeway,either throughpointmutationsor recombination

eventsbetweensimilarsequences.

4.3.3.1.6 Investigatingcph1silencing

Figure 4.28 demonstrates that the three TN72_WUCA2_cph1-HA lines were

homoplasmic,meaningthatallofthe~70chloroplastgenomecopiescontainedthe

desiredexpressioncassette.Thiswasachievedbyseveralroundsofselectionupon

minimal medium, thus selecting for photosynthetically-competent cells and

promoting the presence of the transformed plastome copies. However, once

homoplasmicityhadbeenachievedandconfirmed,cellsweregrownmixotrophically

upon acetate and under light which promotes faster growth. Had the cell been

heteroplasmic at this stage and the GOI was sufficiently toxic, it is possible that

selection could be driven the otherway, back to the photosynthetically deficient

recipientline.However,theselectionforphotosynthesisissostrongthatthisseems

quiteunlikelyandthelossofpsbHfunctionalityalsoconfersphotosensitivity,further

increasingthepressureontheplastometogainpsbHfunction.However,giventhat

thelineswerehomoplasmic,thiscannotbethecase.ThesensitivityofPCRmeans

thatevenifoneplastomecopywerewithouttheexpressioncassette,thisislikelyto

bevisibleinFigure4.28.

Thereareseveralsitesintheinsertedexpressioncassettethatcouldbesubjectto

recombination events within the chloroplast as they are of endogenous origin,

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namelythepsaAexon1promoterand5’UTR,rbcL3’UTRandtrnW.However,the

nativerbcLgeneandthepsaAexon1arerespectively46and47kbpawayfromthe

insertionsite,andthetrnWgeneis61kbpaway,whichwouldbeexpectedtoreduce

recombinationrates,althoughthisrelationshiphasnotbeenconfirmed(Muddetal.

2014). Furthermore,eachof these repeated sequences is relatively small: trnW is

73bp;psaA exon1promoter and5’UTR is 294bp; and rbcL 3’UTR is 407bp. For

context,Muddetal.(2014)suggestthatrepeatsof650bpseparatedby5kbpresults

inefficientrecombination,butnostrictrulehasbeensuggested.Anotheralternative

isthatpointmutationsineitherthemodifiedtrnWgeneorcph1genehaveresulted

infailedtranslationorinstabilityoftheprotein.

Totesttheabovetheories,thecassetteregionwasamplifiedbyPCRfromgenomic

DNAandsequenced(regionsbandcinFigure4.30),asperformedaftertheinitial

confirmation of homoplasmicity. Sequencing confirmed the presence of both the

holin and trnWUCA genes, with nomutations. The psbH.R primer binds inside the

downstreamflankingregionofthetransformationplasmid,and123abindsupstream

of the trnWUCA gene,which therefore suggests that thewhole cassette is present.

However,theFlank1primerliesoutsidethetransformationplasmid,upstreamofthe

cassette,andnoproductcouldbeseenwhenthisprimerwasused.

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Figure4.30Reactionsa-cproducedtheexpectedamplifiedPCRproduct,whiledandefailedtoproduceanyproduct.Thesuccessfulamplificationofa-cshowsthatthecassetteispresentsomewhereinthegenome.ThesuccessofamplificationinFigure4.28(thesameprimersaseabove)suggeststhatthecassettewasoriginallyinthecorrectlocus.However,thefailureofd&eheresuggests that thecassette isno longerpresent in the intendedneutral sitebetween trnE2 and psbH in the chloroplast. The two lanes show two differentTN72_WUCA_cph1-HAtransformants.PrimersequencescanbefoundinAppendixO:ListofPrimers.

It ispossiblethataverysmallnumberofplastomecopiesremainwiththecorrect

cassetteinsertion,butthatthemajorityofcopieshaverecombinedinamannerso

astosilencegeneexpression.However,asmentionedpreviously,thesensitivityof

PCRwouldnormallyenableevenasinglecopytobedetected.Muddetal. (2014)

does note that recombination has been observedwith relatively small repeats in

Chlamydomonas, for example, 216 bp direct repeats in the chlLgene are able to

193

recombine. The most likely explanation is therefore that some combination of

recombinationeventshasledtotherelocationoftheexpressioncassettetoasilent

siteintheplastome,whichwasthenhighlyselectedfor,giventheapparenttoxicity

ofCph-1.


This chapter has examined three strategies to improve Cpl-1 activity against S.

pneumoniae,thefirsttwoofwhichdealwithdirectproteinengineeringandthethird

attemptstoexploitsynergywithanotherprotein,aholin.

4.4.1 Cellwallbindingsitemutagenesis

SixbindingsitemutatedCpl-1proteinswereexpressedinC.reinhardtiitodetectable,

albeitvarying, levels.However,difficultiesarosewhenattemptingtocomparethe

activityoftheseproteinsaslyticenzymesagainstS.pneumoniae.Firstly,thepotential

forimprovementsaroundthemutationsthemselveswillbediscussed,followedbya

discussionofhowtheactivityassaysmightbeadaptedandsuggestionsofpossible

alternativeassays.

It is noted in 4.3.1.1.1 and illustrated in Figure 4.4 that there are three residues

believed to be of key importance in each choline binding site, two tryptophan

residuesandatyrosineresidue.Hermosoetal.(2003)alsosuggestthatalysineis

involved in capping the site and stabilising the phosphate group of phosphoryl-

choline.Thisinvestigationonlyconsideredthetwotryptophanresiduesofeachsite,

simply due to time constraints and the fact that they appear to be closer to the

choline residue. It appears that mutating the tryptophan residues did indeed

negativelyaffect cholinebinding,but that this resulted in reduced lyticactivity. It

would,therefore,beinterestingtoconsiderthetyrosineandlysineineachbinding

siteas theymayenablea subtlermutation tobeproduced.Alanine scanning isa

commonapproachtomutatingresiduesofinterest,asiteliminatestheroleofthe

polarsidechain,however,inthisinstance,thismayresultintoodrasticachangein

polarity.Therefore,amoreconservativemutationmightbeconsidered,forexample,

the mutation of tryptophan to tyrosine. The binding site is clearly sensitive to

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mutationsandhighlyconserved,andthealaninechangesappeartohavebeentoo

severeandresultedinreducedactivity.

Furthermore,theCpl1_W223A-HAconstructwasneversuccessfullysynthesised,for

reasons unknown. It is interesting to note that the Cpl1_W223A_W230A-HA and

Cpl1_W202A_W209A_W223A_W230A-HA,whichbothcontaintheW223Amutation

inadditiontoothers,produceconsistentlydetectablelevelsofproteinaccumulation.

Thisissomewhatunexpected,asW223isnotastrictlyconservedresidueincholine

bindingrepeats(Figure4.5)whileW209andW230are,thoughnoproblemswere

observedwhentheseweremutated.TheissuesaroseafterPCRwhenattemptingto

insert the gene into pJet, a vector from a commercially available PCR cloning kit

(ThermoFischer),enablingbluntendcloningandtransformationofE.coli.NoE.coli

colonies were observed in any of the attempts to produce cpl1_W223A-HA,

suggestingthismutatedgeneiseitherhighlytoxictoE.coliorforsomereasonunable

toligateintothepJetvector.Infutureeitheradifferentintermediatevector,suchas

pGemTEasycouldbeusedortheintermediatestepcouldbeskippedandattemptto

clonedirectlyintopSRSapIusingadigestedPCRproduct.

An interesting and related point of discussion iswhy the inactive choline binding

repeats(p4-p6)remainsohighlyconserved.Astheserepeatsappeartobesterically

unable to bind choline, it would be reasonable to assume that there was little

evolutionarypressure for themtoremainunaltered.The fact that theyremainso

highlyconservedindicatesthattheyalsohavesomeotherimportantfunction.This

maysuggestthatthethree-dimensionalshapeoftheproteinishighlydependenton

theinteractionoftheserepeatsandthatmutationstothemhaveeffectsmorewide-

rangingthatmerelyalteringcholinebinding.

Giventhatinitialattemptstodisruptthecholinebindingsitesappearedtoresultina

reductionofactivity,andthatthep4-p6repeatsarehighlyconserveddespitetheir

purported inactivity, an alternative protein engineering focus could be taken. For

example,theflexibleacidiclinkerthatjoinsthecellwallbindingdomainandcatalytic

domainwouldbeaninterestingsiteforimprovement.Ifitisindeedthecasethatthe

binding domain remains bound to choline in the cell wall, then the number of

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FigureRemoved

peptidoglycancleavagesthatcanbeperformedbythecatalyticdomainislimitedby

thelengthandnatureofthislinker.WhenproducingArtilysins®,Briersetal.(2014)

investigatedtheeffectofincreasinglinkerlengthbetweenthepeptideandendolysin.

Itwasfoundthatantibacterialactivityincreasedinastepwisefashionasthelinker

was extended, then began to plateau at around 18 amino acids in length (Figure

4.31).

Figure4.31L0,L1,L2,L3,andL4correspondto linkeraminoacidsequencesofAGAS (4),AGAGAS (6),AGAGAGAGAS (10),AGAGAGAGAGAGAS (14), andAGAGAGAGAGAGAGAGAS(18).ReproducedfromBriersetal.(2014).

Theacidic linker forCpl-1 comprises residues189-199,withaminoacid sequence

DDEEDDKPKTA.WhilenotdirectlyanalogoustothelinkerinvestigatedbyBriersetal.

(2014), itwouldbe interesting toattempt toextend theCpl-1 linker to see ifany

improvement in activity can be achieved. However, it is unlikely that much

improvementtoactivitycanbegleanedbyextendingtheacidiclinker,asthiswould

haveprobablybeenhighlyselectedforthroughoutevolution.Thisisincontrastto

choline binding which, as discussed previously, appears to act as a tethering

mechanismtopreventaccidentallysisofpotentialfuturehostbacterialcells.

196

DEAEcelluloseactsasacholineanalogueandhasthereforebeenexploitedforion

exchangechromatographytopurifyCpl-1,usingaprotocolfirstdescribedbyLoeffler

etal.(2001).Cpl-1bindstoDEAEandcanthenbeelutedusingafreecholine.This

method was not used in this investigation, as choline binding was the object of

investigation. However, the method could be adapted into an assay for choline

bindingability.Forexample,amutatedCpl-1proteindisplayingweakenedcholine

bindingwouldbeelutedfromtheDEAEcolumnmoreeasily thanstronglybinding

Cpl-1.Thisassaycouldbeusedtoconfirmthatanychangeinactivitywasindeeddue

tocholinebindingalteration,andnotduetosomeotherunknownfactororthree-

dimensionalshapechange.

Theproteinengineeringstepstakeninthissectionwerehighlyspeculativeandwould

have benefitted from testing a larger number of mutants more quickly. For this

reason,thechoiceofC.reinhardtiiasaplatformlimitedtheseinitialinvestigations,

as transformations take more time, recombinant protein yields are lower, and

background antimicrobial effects disrupted activity assays compared to other

platformssuchasE.coli.ThisworkwouldhavebeenbettersuitedtoE.coli,which

enablesrapidprototypingandtestingofrecombinantproteins.C.reinhardtiishould

thenbeusedfurtherdowntheproductionline,oncetheproteinsequencehasbeen

optimised.Atthatpoint,themanybenefitsofusingC.reinhardtiiasaplatformfor

theproductionoftherapeuticrecombinantproteinscanstillbeharnessed.

4.4.2 Cpl-1dimerisation

Cpl-1dimerisationwasattemptedfortworeasons.Firstly,areportbyReschetal.

(2011) demonstrated that pre-dimerised Cpl-1 had a 10-fold reduction in plasma

clearanceandatwo-foldincreaseinactivity.Secondly,theC.reinhardtiichloroplast

isknowntocontrolredoxreactionsandiscapableofcatalysingdisulphidebondsin

recombinant proteins (Tran et al. 2009), which can be further increased by the

additionofselenocystaminetothegrowthmedium(Ferreira-Camargoetal.2015).

However,ourattempttoproduceaCpl-1dimerintheC.reinhardtiichloroplastwas

unsuccessful,aswasinvitroformationofdisulphidebondsusinganE.coliproduced

Cpl-1.

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Themostlikelyreasonforthisfailureisthepresenceofepitopetagsontheprotein

C-terminus,aspreviouslydiscussed.Thefirststeptowardstestingthistheoryisto

redesign the cpl1_dimer gene such that it encodes an N-terminal tag, with no

additionalaminoacidsontheC-terminus.Thegeneshouldalsobetransformedinto

E.coliusingaplasmidsuchaspIN-IIIwhichwilltargettheproteintotheperiplasm

fordisulphidebondcatalysis,orintoanengineeredstrainofcompetentE.colisuch

asRosetta-gami™.IfthissuccessfullyproducesaCpl-1dimer,whichitshouldasitis

asimplereplicationoftheworkbyReschetal.(2011),thenthesamegeneshouldbe

transformedintotheC.reinhardtiichloroplast.Atthispoint,itwillbeclearthatifno

dimercanbeseen in theC. reinhardtii transformants, then it isan issuewith the

platform,ratherthantheprotein.

IfCpl-1dimerscanaccumulateintheC.reinhardtiichloroplast,theninvestigations

intotheuseofselenocystaminecanbeuntaken.Itmaybethatthisstrategycanbe

usedtoincreaseaccumulationlevelsofCpl-1intotal,particularlyifthedimerismore

stable than themonomer. An investigation can also be carried out intowhether

dimerised Cpl-1 is less susceptible to degradation whilst in storage, as it would

potentiallybelessaccessibletoproteasesandlesspronetodenaturation.Thiswould

beanimportantpropertyforthefuturecommercialisationanddistributionofCpl-1

asatherapeutic.

4.4.3 Holinproduction

Holinsenableendolysinstocrossthecellmembraneduringthebacteriophagelytic

cycle. The treatment of some bacteriawith both endolysins and holins has been

showntohaveasynergisticeffectuponcelllysis(Shietal.2012;Songetal.2016).

However,theproductionofholinsinbacterialsystemsisproblematicastheyinsert

intocellmembranesanddepolarisethecell.Herewehavereportedthesuccessful

accumulation of the holin Cph-1 in the C. reinhardtii chloroplast but found that

accumulationquicklyceased.

The‘silencing’ofCph-1isworthyofinvestigation,aschloroplasttransformationsare

not usually susceptible to gene silencing, although it is common in nuclear

transformations.Themost likelyexplanation is that in facthomoplasmicityof the

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plastomewasneverachievedandthatsomenon-specificPCRamplificationoccured,

whichledtoFigure4.28appearingtoconfirmhomoplasmicity.SequencingofthePCR

productcouldhaveconfirmedthisandaTN72negativecontrolshouldhavebeen

includedtodemonstratethatheteroplasmicitywouldhavebeendetected.However,

asdiscussedpreviously,even if thiswas thecase it seemsunlikely that thestrain

would revert to the parental sequence, as loss of psbH function results in

photosensitivity,sogrowthunderbrightlightwouldalsoencouragehomoplasmicity.

Furthermore,aninvestigationbyPCRofthegenomicDNAisdisplayedinFigure4.30,

andclearlydemonstratesthatthecassetteispresentsomewhereintheC.reinhardtii

genome. However, the same investigation failed to confirm, or refute, that the

cassettewas located in the targeted location,namelybetweenthe trnEandpsbH

genesinthechloroplast.AprimerlocatedbeyondthepsbHgeneisrequiredsothat

the entire region can be amplified to confirm that the cassette is not present or

otherwise-thiswouldbeverystraightforward.

We know that over the millennia since the first photosynthetic prokaryote was

engulfedintoaeukaryotetobecomeachloroplast,thevastmajorityofthegenetic

information fromthechloroplastgenomehasbeeneliminatedormigrated to the

nucleus,leavingonlygenesessentialforphotosynthesisinthechloroplast(Howeand

Purton2007).Itispossiblethatthehighlyundesirablecph1-containingcassettehas

been translocated to the nucleus under a similar mechanism, and once there,

silencingmechanismsareprolific.ThepotentialmechanismforsuchaDNArelocation

is not understood, but such a mechanism is known to exist (Cullis et al. 2009).

Furthermore, such transfers have been shown to occur at high frequencies in

tobacco:morethan1in5millioncellswereabletosuccessfullytransferamarker

fromthechloroplasttothenucleus(Stegemannetal.2003).Suchaneventwould

explainthepresenceofthecassette,yetlackofexpression.Amethodsuchasinverse

PCRcouldthenbeusedtodiscoverthelocationofthecassette.

Regardless of the mechanism used by the C. reinhardtii cells to prevent Cph-1

accumulation, it clearly is not tolerated by the cell. It is likely that the holinwas

inserting into the thylakoid membranes, given their similarity to bacterial cell

membranes.AsshowninFigure4.3,alargenumberofholinmonomersarerequired

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toformaraftandthenoligomeriseintoaholebeforeanydepolarisationwilloccur.

Itmaybethattheholinwasonlyweaklyexpressedandthereforenotlethaltothe

cell,butcausedenoughselectivepressurethatitwashighlyundesirable.Thiswould

explainwhytransformantswereobtainedbutquicklylosttheirCph-1expression.

ItislikelythatanyrepeatedattempttoexpressCph-1intheC.reinhardtiichloroplast

will result in the activation of a mechanism to prevent its expression. However,

holins,unlikeendolysins,arenotspeciesspecificinnatureandaregenerallyableto

insertintothemembranesofawiderangeofbacterialspecies(Savvaetal.2008).It

wouldbeinterestingtoexpressaholinandanti-holinpair,suchasS105andS107

fromthelambdabacteriophage,aswellastheholinalone,toseewhetherexpressing

thepairpreventssilencingoftheholingene.Thiswouldenableacontinuationofthe

investigationintowhethertheadditionofaholinalongsideCpl-1canimproveitslytic

activity.

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Chapter5 ExtendingtheChlamydomonasreinhardtiichloroplast

platformtootherendolysins

5.1 Introduction

5.1.1 PreviousendolysinexpressionintheC.reinhardtiichloroplast

DrHenryTaunt,workinginthePurtonLab,wasthefirsttosuccessfullyexpressan

activeendolysinintheC.reinhardtiichloroplast(Taunt2013).Theworkshowedthat

Cpl-1 accumulated to a relatively high level, approximately 1% of total soluble

protein, in the chloroplast and that it was active against S. pneumoniae in vitro.

However,DrTauntwasunabletoshowexpressionoftwootherendolysins,Gp20and

Lys16thattargetPropionibacteriumacnesandStaphylococcusaureus,respectively.

TheworkbyTaunt (2013)alsoattemptedto identifyelementsofendolysingenes

thatresultedinsuccessfulorunsuccessfulexpressionintheC.reinhardtiichloroplast.

However,while several fusion proteinswere created to test this theory, no clear

conclusions could be drawn. Additionally, work by Dr Laura Stoffels, also of the

Purtongroup,achievedexpressionandactivityoftheS.pneumoniaeendolysinPal,

but failed to show activity of theStaphylococcus aureus targeting endolysinφ11,

despitedetectingexpression (Stoffels2015).AnotherendolysinSPN9CC, targeting

Salmonellatyphimurium,wasexpressedbyDrRosieYoungasaproofofconceptthat

recombinantproteinshighlytoxictoE.colicanbesynthesisedintheC.reinhardtii

chloroplast using a synthetic tRNA system, but the activity of SPN9CC was not

investigated(YoungandPurton2015).AsaGram-negativetargetingendolysin,itis

unlikelytobeactiveagainstthetargetbacteriumwithoutmodificationowingtothe

presenceoftheoutermembrane.

Current work in the Purton Group by Juliana de Costa Ramos (unpublished) is

focussed upon producing endolysins targeting Gram-negative bacteria. Two such

endolysins (LysAB2 and LoGT-023) have been successfully expressed in the C.

reinhardtii chloroplast, and although the ‘target bacteria’ for both endolysins is

Acinetobacterbaumannii,Gram-negativeendolysins tend tobemuch less specific

than those targeting Gram-positive bacteria and therefore have a much broader

201

spectrumoflyticactivity.Forexample,theendolysinLysAB2hasbeenreportedto

effectively lyse Staphylococcus aureus, Bacillus subtilis, Streptococcus sanguis,

Escherichia coli, Citrobacter freundii and Salmonella enterica, all in addition to

Acinetobacter baumannii, which the bacteriophage producing LysAB2 specifically

infects(Laietal.2011).Table5.1listsallknownattemptsatexpressingendolysinsin

theC.reinhardtiichloroplasttodate.

Endolysin Targetpathogen SuccessfullyexpressedinC.reinhardtiichloroplast

Activeagainsttarget

pathogen

Reference

Cpl-1 Streptococcuspneumoniae

Yes Yes (Taunt2013)

Gp20 Propionibacteriumacnes

No N/A (Taunt2013)

Lys16 Staphylococcusaureus

No N/A (Taunt2013)

Pal Streptococcuspneumoniae

Yes Yes (Stoffels2015)

φ11 Staphylococcusaureus

Yes No (Stoffels2015)

SPN9CC Salmonellatyphimurium

Yes Nottested (YoungandPurton2015)

LoGT-023(Artilysin®)

Acinetobacterbaumannii

Yes Yes JulianadeCostaRamos(unpublished)

LysAB2 Acinetobacterbaumannii

Yes Yes JulianadeCostaRamos(unpublished)

CD27L1-179 Clostridiumdifficile Yes Yes Thisstudy

Table5.1ThefulllistofendolysinexpressionattemptsbythePurtonGroup.SPN9CC,LoGT-023 and LysAB2 target Gram-negative bacteria,while the others all target Gram-positivebacteria.

5.1.2 Theneedformoreendolysins

Asdiscussedin1.2.2.4,Gram-positiveendolysinshaveaverynarrowspectrumoflytic

activity. This property is highly advantageous in some respects, yet highly

problematicinothers.Theadvantageofanarrowspectrumantibioticisthatonlythe

202

target pathogenic bacteria are killed, while the commensal bacteria remain

unharmed.Thispropertylimitsthedevelopmentofresistanceandtheopportunity

forpathogenicre-colonisation.Thedisadvantageisthatapreciseidentificationofthe

pathogen causing thediseasemust bemadebefore the correct endolysin canbe

prescribedbyaclinician,andadistinctendolysintherapeuticmustbeproducedfor

everycommonbacterialinfection.

Unlike conventional antibiotics, where a single new drug discovery (such as

teixobactin 1.1.2.2) has the potential to treat hundreds of different bacterial

infections, endolysin therapy requires thedevelopment of a large arsenal of very

specificproteinpharmaceuticals.AsshowninTable5.1,theabilitytoproduceactive

endolysinsinC.reinhardtiiisfarfromroutine,withonlya~56%successratesofar.It

isimportant,therefore,tocontinuetoattempttoexpressnewendolysinsaswellas

totrytounderstandthefactorsaffectingexpression.Thischapterdemonstratesthe

successful expression of CD27L in the C. reinhardtii chloroplast, as well as

documentinganattempttoachieveexpressionofGp20,whichhasfailedtoexpress

inpreviousefforts.


1. Toproduceatransgenic lineofC.reinhardtii inwhichtheendolysinCD27L

accumulates.

2. TodemonstratetheactivityofCD27LonC.difficileinvitro.

3. ToproduceatransgeniclineofC.reinhardtiiinwhichGp20accumulates.

4. TodemonstratetheactivityofGp20onP.acnesinvitro.

203


5.3.1 Clostridiumdifficile,ΦCD27andCD27L1-179

ClostridiumdifficileisaGram-positivebacteriumofparticularimportanceduetoits

prevalenceasacommonhospital-acquiredinfection.C.difficile infection(CDI) isa

toxin-mediatedinfection,andisthemajorcauseofpseudomembranouscolitis(Best

et al. 2012). The ability of C. difficile to form spores, which canwithstand harsh

conditionsandantibiotics,meansthat it isoftenthefirstbacteriumtore-colonise

thehumangutaftertreatmentwithbroad-spectrumantibiotics.Whileresistanceto

antibiotics has been reported in C. difficile, vancomycin remains the first line of

treatmentanduntil2014resistancetovancomycinwasrarelyobserved(Hargreaves

andClokie2014).However,recentlytherehavebeenreportsofreducedvancomycin

susceptibility(Spigaglia2016),emphasizingtheneedfornewtreatments.

5.3.2 C.difficileendolysinCD27L

The bacteriophage ΦCD27 infects C. difficile, and its endolysin, CD27L, has been

identifiedanddemonstratedtocauseC.difficile lysis invitrowhenexpressedasa

recombinantproteininE.coli(Mayeretal.2008).CD27Lhasasimilarconformation

tomanyendolysins–aC-terminalcellbindingdomain,andanN-terminalcatalytic

domain. Removal of the C-terminal domain, to create CD27L1-179 was found to

improveenzymeactivity andmarginally extend the lytic rangeof theendolysin –

enablingittolysemorebacterialspecies(Mayeretal.2008).

The C. reinhardtii chloroplast has been successfully used to express two active

endolysinstodate,Cpl-1andPal,bothofwhicharespecifictoS.pneumoniae.To

developtheC.reinhardtiichloroplastasaplatformtoproduceendolysins,andgiven

thespecificnatureofendolysins, it is importanttoshowproductionofendolysins

that are active against a range of bacteria. To this end, the truncated endolysin

CD27L1-179,whichhasapartiallyresolvedcrystalstructure(PDBcode4CU5and3QAY)

andhasbeenshowntobeactiveagainstasmallrangeofbacteriawhenproducedin

E.coli,waschosenasanovelcandidateforC.reinhardtiichloroplastexpression.

204

5.3.2.1 ProductionofaC.reinhardtiilineexpressingCD27L

From hence forth, CD27L will refer to the truncated version of the endolysin,

previouslyreferredtoasCD27L1-179.

5.3.2.1.1 Genedesignandcodonoptimization

TheCD27LproteinisaccessiblebyPDBcodes3QAY(catalyticdomain)and4CU5(Cell

bindingdomain).The179-aminoacidsequence(catalyticdomain)wasobtainedfrom

RCSBPDB(ahistidineaminoacidwasremovedfromtheN-terminus,whichremained

fromtheHis-tag)andthegenesequencewascodonoptimizedfortheC.reinhardtii

chloroplast as described in 2.3.2. AnHA tagwas added to the C-terminus of the

proteincodingsequence forwesternblotdetection,andSapIandSphI restriction

siteswere included for cloningpurposes.The full-lengthgenewas synthesizedby

IntegratedDNATechnologiesasagBlock®GeneFragment(AppendixJ).


GiventhatCD27LhaspreviouslybeenexpressedinE.coliwithoutdetrimentaleffects

toE. coli growth, therewerenoconcernsabout its toxicityduringcloning, so the

pSRSapI transformation vector could be safely used instead of the variant vector

pWUCA2thatallowsUGAstopcodonstobeintroducedintothecodingregion(Young

andPurton2015).ThesynthesizedDNAwasdigestedwithSapIandSphIrestriction

enzymesandligatedintothepSRSapIvectorandtheinsertionwasconfirmedbyDNA

sequencing.


The recipient line TN72 was transformed by the glass bead method using the

pSRSapI_cd27l-HAplasmid.Theresultingtransformedline isrepresented inFigure

5.1.PhotosystemIIfunctionalityisrestoredbypsbHandtheresultingtransformants

werecapableofgrowingphototrophicallyonminimalmedia.Transformantswere

showntoreachahomoplasmicstatebyPCR(AppendixK)asdescribedin2.3.17.

205

Figure5.1TN72recipientstrainsuccessfullytransformedtocarrycd27l-HAunderthepsaAexon1promoter/5’UTRinthechloroplast,andwithpsbHfunctionalityrestored.


WholecellpreparationsofthreeTN72_SR_cd27l-HAtransformants(T1-3),aswellas

TN72_SR_cpl1-HA and TN72_SR_Control (as positive and negative controls,

respectively) were prepared and separated by SDS-PAGE. After blotting to a


probedwith anti-HAprimary antibody, then incubatedwith an IRDye® secondary


resultingimageisshowninFigure5.2.

Figure 5.2 Western blot analysis shows that a ~21 kDa protein is detected by anti-HAantibodiesinthethreeTN72_SR_cd27l-HAextracts,andnotinthenegativecontrol.ThisislikelytobeCD27Lprotein.

(kDa)

206

CD27L protein accumulates to easily detectable levels in all three transformant

clones,andisoftheexpectedsize.

5.3.2.2 ActivityofCD27Lcrudeextractagainst‘stripped’E.coli

SinceitwasnotfeasibletosetupC.difficilecultivationfacilitiesinourlab,anassay

using‘stripped’E.coliwasusedsinceCD27Lhadbeenshowntobeactiveagainstthis

bacterium(Mayeretal.2011).TheE.colihadtobestrippedoftheirouter-membrane

toenableaccesstothecellwallbytheendolysinasdescribedin2.7.2.2(Nakimbugwe

etal.2006).

Crude extracts of TN72_SR_cd27l-HA and TN72_SR_Control were prepared as

describedin2.5.1,andaBradfordassaywasperformedtomeasurethetotalprotein

ineachsample.5μgofeachcrudeextract,aswellas5μgofBSAtoactasanosmotic

pressurecontrol,wereaddedtoa96-wellplate,containing200μlofstrippedE.coli

ineachwell.ThechangeinOD595wasmeasuredbyaFLUOstarOmegaplatereader

(BMGLabtech)overaperiodof200minutesat37°C.TheresultingchangeinOD595

isdisplayedinFigure5.3.

207

Figure 5.3 A turbidity reduction assay,measuring the reduction in OD (595 nm) inwellscontainingE.colistrippedoftheirouter-membraneandchallengedwithcrudeC.reinhardtiiextracts.TheBSAisusedasanosmoticpressurecontrol,whiletheNaPibuffershowstherateofcellautolysiswhenaddedtothecellsintheabsenceofosmoticpressure.

ThecrudeextractcontainingCD27LappearstoshowactivityagainstthestrippedE.

colicells,asthereisamuchgreaterdropinODthanintheBSAcontrol.However,

theredoesappeartobesomeactivityfromthecrudeextractofthecontrolstrain

also.TheshapeofthecurveforthetwoC.reinhardtiicrudeextractslooksmorelike

thecharacteristickillcurveexpected,thanthelinearrateobservedfortheaddition

ofNaPiBuffer.

5.3.2.3 ActivityofCD27LcrudeextractagainstC.difficile

InordertotesttheendolysinagainstC.difficile,acategorytwobiohazardforwhich

ourlabwasnotequipped,acollaborationwasestablishedwithDrMelindaMayer,

whosegroupattheQuadramInstituteBioscience(formerlytheNorwichInstituteof

208

FoodResearch)werethefirsttoidentifyCD27L(Mayeretal.2008).Thisenabledthe

testing of the CD27L-containing crudeC. reinhardtii extract directly onC. difficile

usingtheMayergroup’sestablishedprotocol.

A turbidity reduction assay was performed as described in 2.7.2.3. Two

concentrationsof crudeextract, inNaPibuffer,wereused: 10μgand50μg. The

purifiedCD27LwasprovidedbyDrMayerandusedasapositivecontrol.Itwasan

old(2008)NiNTA-purifiedCD27LsampleproducedinE.coli,sotheconcentrationwas

deemed irrelevant due to its age as much of it was thought to have degraded,

however,itstillservesasausefulpositivecontrol.

Figure5.4Clostridiumdifficileturbidityreductionassay.ThepurifiedCD27LwasprovidedbyDrMayerasapositivecontrolandTN72_SR_Controlasanegativecontrol.PerformedbyDrMelindaMayeratQuadramInstituteBioscience,Norwich,UK.

209

Figure5.4showsthattheTN72_SR_Control,whichwasusedasanegativecontrol,

appearstohaveaninhibitoryeffectuponcelllysiswhencomparedtotheNaPibuffer

control.Thisisbelievedtobeduetotheeffectofahigherosmoticpressureonthe

C.difficilecellsduetothepresenceofotherproteinsinthecrudeextract,andhas

beenreportedpreviously(Mayeretal.2010).

5.3.2.4 EnrichedCD27Lcrudeextract

TheosmoticpressureeffectsseenintheC.difficileturbidityreductionassay(Figure

5.4),andtheapparentactivityofthecontrolextractobservedinFigure5.3against

strippedE.colicells,hinderedourabilitytosatisfactorilymeasuretheactivityofthe

C. reinhardtiiproducedendolysin.Consequently, itwasdecidedtochangetheHA

epitopetagontheC-terminusoftheCD27LproteintoaStrepIItagtobetterenable

enrichmentor purification. Thiswasperformed to enable abetter comparisonof

endolysinactivitieswithouttheeffectsoftheC.reinhardtiicrudeextract.

5.3.2.4.1 ExpressionofCD27L-StrepIIinC.reinhardtii

PCRwasusedtochangetheepitopetagonthecd27lgene.Thereverseprimerwas

designedtohaveanoverhangwiththeStrepIItagsequenceattached,thusremoving

theHAtagandaddingtheStrepIItag.TheStrepIItagisshowninlowercase,andthe

startandstopcodonsareunderlinedontheprimersbelow:

cd27l.F: TGGTATTGCTCTTCAATGAAAATTTGTATTACT

cd27l_StrepII.R: ACTGACGCATGCTTATTAtttttcgaattgtgggtgagacca GTTAATGTTTTTGTTTAAAACACCTTCAAC

ThePCRproductwasdigestedwithSphIandSapIrestrictionenzymesandligatedinto

pSRSapIasdescribedin2.3.10and2.3.11.Sequencingconfirmedtheadditionofthe

correctencodingsequencefortheStrepIItag.

The recipient line TN72 was transformed by the glass bead method using the

pSRSapI_cd27l-StrepII plasmid. The resulting transformed line is represented in

Figure 5.5. Photosystem II functionality was restored by psbH and the resulting

transformants were capable of growing phototrophically on minimal media.

210

Transformantswere shown to reachahomoplasmic state (Appendix L)byPCRas

describedin2.3.17.

Figure5.5 TN72 recipient strain successfully transformed to carrycd27l-StrepII under thepsaAexon1promoter/5’UTRinthechloroplast,andwithpsbHfunctionalityrestored.

5.3.2.4.2 ConfirmationbyWesternblotting

WholecellpreparationsofTN72_SR_cd27l-StrepII,aswellasTN72_SR_cd27l-HA,and

TN72_SR_Control(aspositiveandnegativecontrolsrespectively)werepreparedand

separatedbySDS-PAGE.Afterblottingtonitrocellulosemembraneandblockingas

described in2.4.5, themembranewasprobedwith anti-StrepII primary antibody,

thenincubatedwithIRDye®secondaryantibody.Fluorescencewasobservedusing

the LiCorOdyssey® CLx system. Themembranewas then re-probedwith anti-HA

primary antibody and IRDye® secondary antibody. The two images are shown in

Figure5.6.

211

Figure5.6Westernblotanalysisof themembraneprobedfirstlywithanti-StrepIIprimaryantibodies(left),thenadditionallywithanti-HAantibodies(right).CD27Laccumulationwasdetected in the expected cell lines i.e. CD27L-StrepII was detected with the anti-StrepIIantibodyandtheuseoftheanti-HAantibodyalsorevealedthepresenceofCD27L-HA.Thenon-specificbandconfirmsthatasimilarquantityofextractwasloadedontothegelforallthreestrains.

5.3.2.4.3 Relativeproteinaccumulationlevels

The use of the protein standard, described in 3.3.1.2.5, enabled the relative

quantificationofCD27LproteinaccumulationinthetwoC.reinhardtii lines.Figure

5.7showscomparisonsofthetwowholeproteincellpreparationsusedin5.3.2.4.2

anda10μgproteinstandardisincluded.ThestandardistaggedwithHAandStrepII

epitopes,enablinganapproximatecomparisonoftheproteinaccumulationseenin

eachsample.

(kDa)

212

Figure5.7Westernblotanalysisofwholecellextracts,firstnormalisedbycultureOD740andcompared to a 10 μg protein standard. Quantification was performed using the LiCorOdyssey®CLxsystem,afterwhichthestandardwasusedtonormalisethetwoblotsandthesignalfromthe21kDabandscomparedinabarchart.

The protein accumulation level of the HA-tagged protein appears to be

approximately six times greater than the StrepII-tagged protein. However, as

discussed in 3.3.2.2.4.3, the difference observed is possibly due to the different

accessibility of the epitope tags on the Cpl_1-HA-StrepII protein standard. This is

supportedbythatfactthatthispatternalsoappearsbetweentheCpl1-HAandCpl1-

StrepIIaccumulationlevelsanditseemsunlikelythattheStrepIItagishavingsucha

consistentlydetrimentaleffectuponaccumulationlevels.

(kDa)

213

5.3.2.4.4 EnrichmentofCD27L-StrepIIusingHiTrapStrepIIColumn

TheinclusionofaStrepIItagonCD27Lenablesenrichmentoftheproteinandwill

thusallowaclearerinvestigationintoitsantibacterialproperties.C.reinhardtiicell

extractswerepreparedasdescribedin2.5.1,andultracentrifugedat100,000gfor1

hourtoremovecelldebris.Theresultingultracentrifuged(UC)supernatantwasthen

enrichedforStrepII-taggedproteinsusingthecommerciallyavailableHiTrapStrepII

Column, as described in 2.5.3.Western blot analysis of the resulting fractions is

showninFigure5.8.

Figure5.8Westernblotanalysisusinganti-StrepIIprimaryantibodyandIRDye®secondaryantibody.FractionsfromsamplepreparationandtheHiTrapStrepIIcolumnwerecollectedandseparatedbySDS-PAGE.Theelutedfractionhasnonon-specificband,and ‘Elution2’wasusedforfurtherexperiments.

ItisclearfromFigure5.8thatenrichmenthasbeensuccessful,andthattheeluted

fractionscontainhighquantitiesofCD27Landlowquantitiesofthenon-specifically

recognisedprotein.ItisalsonoticeablethatalargeproportionoftheCD27Lprotein

islostintheultracentrifugedpellet,butforthepurposesofthisinvestigation,thisis

not important. In the future,optimisationof thisprotocolmay result inagreater

yield.

5.3.2.4.5 ActivityofenrichedCD27LcrudeextractagainststrippedE.coli

TheenrichedCD27Lextractwaspreparedasabove(5.3.2.4.4)andanenrichedCpl-1

extractwasusedasanegativecontrol.ABradfordassaywasperformedtomeasure

thetotalproteinineachsample.

214

TheenrichedextractwasinitiallytestedforactivityusingthestrippedE.coliassay.

0.4μgofeachenrichedextract,aswellas5unitsofeggwhitelysozymetoactasa

positivecontrol,wereaddedtoa96-wellplatewithstrippedE.coli.Thechangein

OD595wasmeasuredoveraperiodof90minutesat37°C.Theresultingchangein

OD595isdisplayedinFigure5.3.

Figure5.9Aturbidityreductionassay,measuringthereductioninOD595inwellscontainingE.colistrippedoftheirouter-membraneandtreatedwithenrichedC.reinhardtiiextracts.TheCpl-1extractactsasanegativecontrol.Onlythefirst25minutesareshown,astheODremainsstablebeyondthis.

TheenrichedCD27Lappearstobehighlyactive,withtheOD595dropplateauingafter

approximately15minutes.TheCpl-1enrichedextractdemonstratesthespecificity

ofendolysins,andactsasanegativecontrolinthisinstance.The20mMNaPibuffer

doesnotappear toproduce thesame levelof cell lysisasobserved inFigure5.3,

215

probablybecausesmallersamplevolumeswereusedinthisinstance–around5μl

asopposedto~30μl.ThereislittledifferenceobservedbetweentheCpl-1andNaPi

buffer, suggesting that this enrichment has removedmany of the other proteins

contributing to the osmotic pressure effect described previously. The lysozyme,

whichwasincludedtoactasapositivecontrol,doescausesomelevelofcelllysisbut

plateauswithanOD595dropofapproximately0.1.

5.3.2.5 ActivityofenrichedCD27LcrudeextractagainstC.difficile

GiventhesuccessofenrichedCD27LagainstE.colishowninFigure5.9,similarfresh

samples were sent to Norwich for in vitro testing upon C. difficile. The turbidity

reductionassaywasperformedasdescribedin2.7.2.3.

216

Figure5.10Aturbidityreductionassay,measuringthereductioninOD600inwellscontainingC.difficilechallengedwithenrichedC.reinhardtiiextracts.TheCpl-1extractactsasanegativecontrol.Thebufferused istheStrep-Tactin®elutionbufferprovidedbytheHiTrapStrepIIcolumnkit,containingdesthiobiotin(abiotinanalogue).0.3μgofeachenrichedextractwereused. TheCD27L (E. coli) refers to apurifiedCD27L recombinantproteinproducedbyDrMayer in E. coli, to act here as a positive control. This turbidity reduction assay wasperformedbyDrMelindaMayeratQuadramInstituteBioscience,Norwich,UK.

The data show convincing lysis of C. difficile cells in vitro, despite the fact that

autolysis appears to be relatively high, as is characteristic of C. difficile when

incubatedforextendedperiodsoftimeinoxygenatedconditions,asC.difficileisan

obligateanaerobe(Mayeretal.2011).Thesampleswere,unfortunately,muchmore

dilutethanDrMayercommonlyusesforturbidityreductionassays,andinthefuture

atechniquesuchasammoniumsulphateprecipitationmayenablemoreendolysin

(~10μgratherthan0.3µg)tobeaddedtoeachwell.

217

TheconclusionsofalloftheseCD27Lexperimentsisthatanotherbiologicallyactive

andstableendolysincanbeproducedintheC.reinhardtiichloroplast.Theaddition

of a StrepII tag to the recombinant protein enables the fast and relatively cheap

enrichmentofcrudeextracts,whichinturnhasallowedustoobservemoreclearly

theactivityoftheendolysin.

218

5.3.3 Propionibacteriumacnes,bacteriophagePA6anditsendolysinGp20

Propionibacteriumacnesisaprevalent,Gram-positive,commensalbacteriumknown

bestforitsroleinthecommonskinconditionacnevulgaris.Acnevulgarisisbelieved

toaffectover80%ofadolescentsandassuch,antibioticstotreatithavebeenover-

usedandresistancehasbecomecommonplace(Leydenetal.1983;BojarandHolland

2004;Tayloretal.2014).Somebroad-spectrumantimicrobialagents,suchasbenzoyl

peroxide, while still effective, have side effects that make their prolonged use

unadvisable(Fultonetal.1974;Bojaretal.1995).

Thetreatmentofacnevulgarisoffersalargemarketandunmetneed,whichcould

possiblybeservedbyendolysintreatment.Asacnevulgarisisatopicalaffliction,it

alsopresentsaplausibletargetfortreatmentwithaC.reinhardtiicrudeextract.

The bacteriophage PA6 is known to infect P. acnes and in 2007 its genomewas

sequenced(Farraretal.2007).ThesamestudyshowedthatPA6wasabletoinfect

32differentP.acnesisolatesincludingantibioticresistantstrains.PA6belongstothe

Siphoviridae, the same virus family as Dp-1 (from which the endolysin Pal was

isolated)andΦ11(fromwhichaStaphylococcalendolysinwasisolated).Theresulting

genomeanalysis identifiedageneencodingaputativeamidase,namedgp20.The

identificationofgp20asencodinganendolysinisfurthersupportedbythefactthat

thegeneimmediatelydownstreamofit,gp21,appearstoencodeaholin:aclassic

endolysin-holingenearrangement.

Todate,therearenoreportsintheliteraturedescribingthesuccessfulexpressionof

Gp20asarecombinantprotein,orademonstrationofpeptidoglycancleavageofP.

acnesorcelllysis.However,aUSpatent(US_2012_0195872_A1)wasfiledin2010by

Lysando(anaforementionedendolysincompany)describingthefusionofGp20toan

18aminoacidpeptidesequence,whichwasshowntobeactiveagainsttwoP.acnes

strains, presumablyworking in a similarmanner to theArtilysins®, alsodiscussed

earlier(1.2.2.1).

219

5.3.3.1 PreviousattemptstoexpressGp20intheC.reinhardtiichloroplast

SeveralpreviousattemptstoexpressGp20intheC.reinhardtiichloroplasthavebeen

madeinthePurtonlab.Thesehaveincludedinsertionintothechloroplastindifferent

expressioncassettes,withdifferentpromoter/5’UTRs,andevenaschimericgenes,

butdetectionhasnotbeenachieved(Taunt2013).

5.3.3.2 NewattempttoproduceaC.reinhardtiilineexpressinggp20

Onevariablethathasnotbeenalteredhowever is theC-terminalHAtag.Epitope

tags,suchastheHAtag,areanextremelyusefultoolfordetectingproteinsforwhich

noantibodyisavailable,suchasnewproteins.Traditionallyepitopetagsareadded

toaproteinterminusinthehopethattheywillnotinterferewithproteinfunction.

However, as seen in this thesis and elsewhere in the literature (e.g. Brault et al.

(1999)), epitope choice and position can alter protein accumulation levels and

function.Aterminaltagremainedasensiblechoice,giventhatthestructureofthe

Gp20proteinisunknown,soasimpleswitchfromtheC-terminustotheN-terminus

oftheproteinwasproposed.

5.3.3.2.1 Genedesignandcodonoptimizationofgp20N

The success of the pWUCA2 expression vector (which allows stop codons to be

insertedintoagene-of-interest,therebycircumventingtoxicityissueswhencloning

inE.coli(YoungandPurton2015))inachievingsimilarlevelsofproteinaccumulation

intheC.reinhardtiichloroplast,andtheunknowntoxicityoftheGp20endolysininE.

coliledustousethepWUCA2plasmidasaprecaution.CodonUsageOptimizerBeta

0.92(Kong2013)wasusedtooptimizethegp20geneforC.reinhardtiichloroplast

expression. Three tryptophan codons spaced throughout the genewere changed

fromUGGtoUGA.TheHAtagcodingsequencewasincludedattheNterminus.SapI

andSphIrestrictionsiteswereincludedforcloningintothepWUCA2vector.Thefull-

lengthgp20N-HA(the‘N’indicatesthattheHAtagisplacedontheNterminus)gene

wassynthesizedbyIntegratedDNATechnologies(AppendixM).


SapI and SphI restriction enzymes were used to digest and ligate the gene into

pWUCA2inframe,producingtheplasmidpWUCA2_gp20N-HA.DH5αcompetentE.

220

colicellsweretransformedwiththeresultingplasmidandsequencingconfirmedthe

correctinsertion.


ThepWUCA2_gp20N-HAplasmidwasusedtotransformrecipientC.reinhardtiistrain

TN72 via the glass bead method as described 2.3.13. Figure 5.11 illustrates the

resultingC.reinhardtiicellline,pWUCA2_gp20N-HA.

Figure5.11TN72recipientstrainsuccessfullytransformedtocarrygp20N-HAunderthepsaAexon1promoter/5’UTRinthechloroplast,andwithpsbHfunctionalityrestored.AmodifiedtrnWgenehasbeenco-introducedtotranslatetheintroducedUGAcodons(indicatedasredbars)inthegp20Ngeneastryptophans.

TheresultingtransformantswereconfirmedforhomoplasmicitybyPCRasdescribed

in 2.3.17 (Appendix N). Finally, the correct insertion was confirmed by DNA

sequencingasin2.3.18.

5.3.3.2.4 Westernblottingunabletoconfirmaccumulation

To test for the accumulation ofGp20 inC. reinhardtii,whole cell preparations of

TN72_WUCA2_gp20N-HA,TN72_A_gp20-HA(Taunt2013),andTN72_SR_Control(as

twonegativecontrols)werepreparedandseparatedbySDS-PAGE.Afterblottingto


probed with anti-HA primary antibody, then incubated with IRDye® secondary


resultingimageisshowninFigure5.12.

221

Figure5.12Westernblotanalysisofwholecellextracts.TN72_SR_Controlactsasanegativecontrol,andTN72_A_gp20-HAdemonstratesthepreviousattemptmadebyTaunt(2013)toexpressgp20intheC.reinhardtiichloroplast.

Noproteincanbeseen inFigure5.12at35kDa, theexpectedsize forGp20.The

possiblereasonsforwhicharediscussedinthefollowingsection.

5.3.3.3 Possiblecausesforgp20expressionfailure

Asalludedtoearlierinthisthesis,failureofrecombinantproteinexpressionisquite

commonplaceintheC.reinhardtiichloroplastastheplatformisstillintherelatively

early stagesofdevelopment. Twopossible reasons for failure todetectGp20are

eitherthefailureoftranslationorinstabilityoftheprotein.Thissameconclusionwas

drawnbyTaunt(2013),whengp20failedtoexpressinC.reinhardtiionaprevious

occasion, with transcription issues and protein degradation being considered as

unlikelyduetopreviousliteraturesuggestingthattranscriptsareusuallypresentin

failed recombinant protein expression attempts (Rochaix 2001), and endolysins

having evolved to remain stable in a prokaryotic environment (Oey et al. 2009).

SignificanteffortsweremadebyTaunt(2013)toinvestigateandamelioratefailures

of translation.Theseeffortswere focussedupontheribosomebindingsite.Given

thattheupstreamatpApromoter/5’UTRusedbythepASapIexpressioncassetteare

known to be functional (See Cpl-1 work 3.3.2.2.1.4), it was supposed that the

downstreamboxwas playing an important role and in the instanceofgp20,was

hinderingribosomebinding.Thiswasinvestigatedbyincludingthefirst34codonsof

(kDa)

222

theendogenousatpAgenefollowedbytheGOI,tocreateafusion(separatedbya

stromalprocessingpeptidase).TheplasmidusedtoperformthiswasnamedpASap2

andwascreatedbyDrChloeEconomou(Taunt2013).However, thisstrategyalso

failedtoproduceanyvisiblelevelofproteinaccumulation.

5.3.3.4 ProductionofanE.colilineexpressingGp20

The failure to detect Gp20 accumulation in the C. reinhardtii chloroplast after

attempting several strategies led us to investigatewhether Gp20was stable and

activewhenexpressedasarecombinantproteininE.coli.Apreviousinvestigation

byTaunt(2013)failedtoobserveexpressionofGp20inE.colilinestransformedwith

pASapI containing the gp20 gene, believing a lack of optimisation of the atpA

promoter/5’UTRforE.coliexpressiontoberesponsible(Wannathongetal.2016).

Weinvestigatethisfurtherhere,asifproteinaccumulationcanbeachievedinE.coli,

then it suggests that the protein is likely to be stable in C. reinhardtii and that

translation is likely to be the key issue in the failure to detect the recombinant

protein.


Thegp20N-HAgeneusedpreviously couldnotbe reused for expression inE. coli

owingtothepresenceofstopcodonsinthegene,asitwasoriginallydesignedfor

use with the pWUCA2 plasmid. A gp20 gene with a C-terminal HA tag, codon

optimized for Synechocystis expression in another project and synthesized by

Integrated DNA technologies, was used as a template. Primerswere designed to

introduceaNcoIrestrictionsiteupstreamoftheATG.ABamHIsitealreadyexisted

below the stop codons so did not needmodifying. The introducedmutations are

showninlowercase,whilethestartandstopcodonsareunderlined.

gp20_NcoI.F: CGTATACccATGGTGCGTTA

gp20.R: GATTGCGGATCCTTATTAGGC

ThePCRproductwasdigestedandligatedintothehighexpression,IPTGinducible,

plasmidpProEXHTb(Figure5.13)andtransformedintoE.colistrainDH5a.

223

FigureRemoved

Figure 5.13 Structural plasmid map of pProEX HTb and details of the multiple cloning site. Reproduced fromhttp://www.biofeng.com/zaiti/dachang/pProEXHTb.html.

224

5.3.3.4.2 Coomassiestainconfirmationofexpression

SynthesisoftherecombinantproteinwasinducedusingIPTGasdescribedin2.6.1.

E. coli lysateswere then createdusingBugBuster™asdescribed in2.6.2 forboth

inducedanduninducedculturesandpreparedforproteinanalysis.Thepreparations

wereseparatedbySDS-PAGEandstainedwithCoomassieBrilliantBlueasdescribed

in2.4.4.ThestainedgelwasanalysedusingtheLiCorOdyssey®CLxsystemandthe

resultingimageisdisplayedinFigure5.14.

Figure5.14WholecellpreparationsofinducedanduninducedE.colitransformedwith

pProEX_gp20-HAwereseparatedbySDS-PAGEandtotalproteinstainingwasperformed

usingaCoomassiestain.

HighaccumulationisobservedintheinducedE.coliattheexpectedsizeforGp20,

whichis~35kDa.Thereisnoproteinseenintheuninducedsampleswhichgivesus

confidencethattheobservedproteinisindeedGp20.

5.3.3.5 ActivityofGp20

AcrudelysateoftheinducedE.coliexpressinggp20wasproducedusingaStansted

‘Pressure cell’ homogeniser (2.6.3), and centrifuged at 5,000 g for 20minutes to

remove cell debris. The lysate was then filter sterilized using a 0.22 μm filter to

remove any remaining viable E. coli cells. The same procedure was applied to a

culture of untransformedDH5α cells as a negative control. UntransformedDH5α

wereusedinsteadofuninducedDH5α_ProEX_gp20-HAtoavoidissuesaroundleaky

expressionofgp20intheuninducedcontrol.

25

32

46

Induced Uninduced

gp20(35kDa)

x10

x5 x1 x10

x5 x1

225

5.3.3.5.1 Crudelysateforactivityassay

Thepellet, lysateand filter sterilized lysatewerepreparedandseparatedbySDS-

PAGE.Afterblottingtonitrocellulosemembraneandblockingasdescribedin2.4.5,

the membrane was probed with anti-HA primary antibody, then incubated with

IRDye®secondaryantibody.FluorescencewasobservedusingtheLiCorOdyssey®CLx

system.TheresultingimageisshowninFigure5.15.

Figure5.15WesternblotanalysisshowsHA-taggedGp20inallthreelanesoftheinduced

DH5α_ProEX_gp20-HAlysate,andnotinthenegativecontrollanes.

AlthoughitappearsthatalargeproportionofGp20proteinislostinthepellet,there

isstilladetectablequantityinthefiltersterilisedlysate.Thisisanencouragingresult,

asitsuggeststhatrecombinantGp20canexistinastableform.Thisfindingreduces

thelikelihoodthatGp20instabilityisthemajorcauseofthelackofdetectableprotein

the C. reinhardtii chloroplast, and requires us to look once again at translation

elongationasthemostlikelyissuepreventingaccumulation.

5.3.3.5.2 ColonyformingunitassayagainstP.acnes

InordertotestwhethertherecombinantGp20isactive,acolonyformingunitassay

againstP.acneswasperformedusingthefiltersterilizedlysatesasshowninFigure

5.15.

(kDa)

226

CulturesofP.acnesweregrownanaerobicallyasdescribedin2.1.5.3.Thecultures

werecentrifugedat5,000gfor20minutesandresuspendedin20mMNaPibuffer.

50 μl of bacterial suspension and 50 μl of filter sterilized lysate or buffer were

incubatedat37°Cfor0,30,60,90,120or150minutes.Three10μlsampleswere

taken after the stated incubation time from each combination and spotted onto

blood agar plates. After anaerobic incubation at 37 °C for oneweek, growthwas

visibleandtheplateswerephotographed,showninFigure5.16.

Figure5.16P.acnesgrowthonbloodagarplatesafterincubationwithcrudeE.colilysateswithandwithoutGp20,orjustwith20mMNaPibuffer.AsisoftenthecasewithP.acnes,individualcoloniestendnottobevisibleandasmalllawnofcellsappearsinstead(Stoffels

2015).

Thepresenceofalawn,ratherthandistinctcoloniesofP.acnesmakesquantification

of the antimicrobial effect of Gp20 difficult to measure. However, visually there

appeared to be no difference in growth between any of the spots regardless of

substrateandincubationtime.Infact,thebufferaloneappearedtoreducethelawn

227

themost,perhapsduetotheosmoticpressureeffectsdescribedpreviouslyinthis

thesis. Unfortunately, this experiment lacks a positive control, without whichwe

cannotbesurethatGp20isnotworkingas,forexample,thevisiblebacteriamaybe

somethingotherthanP.acnesorthedosagemightnotbehighenough.

ThisresulttentativelysuggeststhatGp20,whenproducedasarecombinantprotein

inE.coli,doesnotdisplaystrongantibacterialpropertiestowardsP.acnes,however,

as mentioned, the assay requires improvements for this conclusion to be more

convincing.SinceTaunt(2013)initiallyinvestigatedgp20expressioninC.reinhardtii,

13 other P. acnes targeting endolysins have been identified from other

bacteriophages(Marinellietal.2012).Theseputativeendolysinsdisplayremarkable

sequence similarity, as do the whole bacteriophage genomes themselves. This is

unusualforbacteriophageandunexpected,asusuallyahighlevelofgeneticdiversity

exists amongst bacteriophage (Pride et al. 2012). The differences that do occur

betweenendolysinsequencesaremostlyintheflexiblelinkerregionseparatingthe

twodomains.ThissignificantlevelofsimilarityledMarinellietal.(2012)tosuggest

that any of the 14 endolysins chosen for recombinant production and endolysin

therapywouldbesuccessful.

228


This chapter has attempted to express two endolysins in the C. reinhardtii

chloroplast. The first, CD27L, has been shown in the literature tobe activewhen

producedinE.coliandtestedinvitroagainstC.difficile(Mayeretal.2008).Herewe

haveshownthatCD27LcanbeexpressedinC.reinhardtiitoarelativelyhighlevel

and is active in vitro against both strippedE. coli andC. difficile. In contrast, the

secondendolysin,Gp20,hasnoliteraturedemonstratingitsrecombinantsynthesis

oractivityinvitroorotherwise,andpreviousattemptstoexpressgp20inE.coliand

C.reinhardtiihavefailed,forunknownreasons(Taunt2013).Afterfailingtoachieve

expressioninC.reinhardtiiwithadifferentepitopetagposition,thefocusshiftedto

achievingexpressioninE.coliandassayingforactivityagainstP.acnes.Expressionin

E.coliwassuccessfulbutactivitywasnotsatisfactorilyassayed,aswillbediscussed

furtherbelow.

5.4.1 CD27Lendolysin

TheinvestigationintoexpressingCD27LinC.reinhardtiihasbeenrelativelysuccessful

todate.Expressionhasbeenachievedand theuseof theStrepII taghasenabled

enrichmentofCD27Lfromcrudeextracts,allowingmorepreciseactivityassaystobe

performed.Activityhasbeenconfirmed invitroagainstbothC.difficile, itsnatural

target,andE.colicellswhenstrippedoftheirouter-membrane,whichactsasauseful

assaywhenC.difficilefacilitiesareunavailable.

AswithCpl-1inChapter3,attemptstoquantifyCD27Laccumulationhaveproduced

varied results. The StrepII-tagged CD27L protein appears to accumulate to

significantlylowerlevelsthantheHA-taggedversion–apatternalsoseenwithCpl-

1.The finding thatStrepII-taggedproteinsconsistentlyappear toaccumulate toa

lowerlevelthantheirHA-taggedcounterpartshasledustoquestionthevalidityof

thedouble-taggedproteinstandardproducedin3.3.1.2.6,ashasbeendiscussedin

more detail in 3.3.2.2.4.3. For both this CD27L work and the Cpl-1 work, it is

important toaddress this issueand finda suitablemethodofquantifyingprotein

229

accumulation,suchasraisingantibodiestoCD27Litselfandusingthepurifiedprotein

asastandard.

WhiletheresultsshowninFigure5.10appearcredible,demonstratingtheactivityof

C.reinhardtii-producedCD27LagainstC.difficilerequiresafewminormodifications

totheassaytomakeitmoreconvincing.Thefirstistoperformtheassaywithhigher

concentrationsofendolysin.DrMayerusuallyincludes10μgofendolysinperwell,

whilewewere only able to use 0.3 μg perwell due to the diluted nature of the

extract. This can be easily remedied by performing an ammonium sulphate

precipitationupon theextract to concentrate theprotein. It is expected that this

improvementwillwidenthegapbetweenthepositiveandnegativecontrols,thus

makingtheassaymoreconvincing.Secondly,adistinctionmustbemadebetween

celllysisandcellviability.Whileitisreasonabletoassumethatthetwoarerelated,

aminorreductioninturbiditydoesnotnecessarilymeanthatcellviabilityisequally

unaffected.Forexample,anendolysinmaycleavethepeptidoglycaninonlyafew

locations,enoughtolysethecell,butleavingthewallotherwisemostlyintact.This

willregisterasalowlevelofturbidityreduction,buthasreducedcellviabilitytothe

samelevelasanendolysinthatbreaksthewallintomanypieces.Inordertoconfirm

theefficacyofanendolysin,itisthereforeimportanttoincludeadirectmeasurement

ofcellviability,suchasaCFUassay.Byplatingthecontentsofthewellsfromthe

turbidity reduction assay onto nutrient agar it would be possible to quantify the

remainingnumberofviablebacteria.

Arecentlydiscoveredpropertyofsomeendolysins,suchasCD27LandCTP1L(Dunne

etal.2016)andLys170(Proençaetal.2015),istheirencodingofasecond,internal,

translation start site. This second translation start site results in the additional

synthesisoftheC-terminaldomaini.e.afreecellwallbindingdomain.ThisfreeCBD

formsamultimericendolysinwiththefull-lengthendolysintoenableittobecome

fullyactive.ItwasnotedbyDunneetal.(2016)thattherecombinantendolysinCTP1L

produced froma codon-optimised genedisplayed significantly lower activity than

thatproducedfromthewild-typenucleotidesequence.Thecodonoptimisedversion

produced much lower levels of free CBD, and the addition of recombinantly

synthesisedfreeCBDrescuedactivitylevels.

230

Thecd27lgeneexpressedhereinC.reinhardtiiisthetruncatedversionofthefull-

lengthgene,asdiscussed in5.3.2.1,andassuchdoesnotcontain theCBDor the

putativeinternalribosomebindingsiteidentifiedbyDunneetal.(2016).Dunneetal.

(2016) noted that the free CBD of CD27L was not necessary for activity, but

postulated that it may play a role when the endolysin is under physiological

conditionswhichhaveyettobeinvestigated.

Itwouldbeinterestingtoexpressthefull-lengthCD27Lendolysin inC.reinhardtii,

usingacodonoptimisedversionbutensuringthattheShineDalgarno(SD)andRBS

oftheputative internal translationsiteremainunchanged.Thiswouldanswerthe

questionofwhethertheC.reinhardtiichloroplastmachineryisabletorecognisethis

internaltranslationsite.Itseemsunlikelythattheinternalsitewillberecognisedby

the chloroplast machinery, as although the chloroplast system does share many

featureswithitsprokaryoticancestors,translationinitiationislesswellunderstood

in the C. reinhardtii chloroplast. For example, although chloroplast 16S rRNA

molecules display a highly conserved anti-SD sequence near their 3’ end, the

complementary SD sequences on chloroplast mRNA UTRs are hypervariable, if

presentatall(Rochaixetal.1998).AC-terminalStrepIItagonthefull-lengthCD27L

proteinwillenableenrichmentanddetectionofboththefull-lengthproteinandany

productsynthesisedfromtheinternaltranslationsite.Iftheinternaltranslationsite

fails to produce a freeCBD, then itwould alsobeof interest to express theCBD

independently,underanalternativepromoter/5’UTR.

TheinvestigationbyDunneetal.(2016)concludedthatthefreeCBDoligomerised

withthefull-lengthCD27L,butforthisendolysinitdidnotappeartogreatlyaffect

activity. Therefore, it may be more relevant to look at CTP1L, an endolysin that

targetsClostridiumtyrobutyricum,andwasshowntorequirethefreeCBDforactivity.

Thepotential forCTP1Las a future candidate forC. reinhardtii expressionwill be

discussedlaterinthischapter.

Usually,atthisstageofanendolysinproject,mostfurtherworkwouldinvolvethe

useofananimalmodelforinvivotesting.However,inthecaseofC.difficilemodels,

themosthelpfulnext stage isprobablymoreadvanced in vitro testing,using, for

231

example,atriple-stagechemostatmodelofthehumangut.Thereasonforthisisthat

the most well-used animal model for CDI, the Syrian Hamster, is not fully

standardised and presents several important and problematic differences in

pathophysiologytohumaninfection(Bestetal.2012).Thetriple-stagehumangut

modelwasdesignedbyMacfarlaneetal.(1998)andhassincebeenimprovedand

updated,butessentiallyconsistsofthreeconnected,pHcontrolled,anaerobically-

maintainedvessels,withgrowthmediafedintothetopataspecificflowrate.The

model emulates the characteristics (spatial, temporal, nutritional and

physicochemical)oftheproximaltothedistalbowel (Bestetal.2012).Thetriple-

stagechemostathumangutmodelhasbeenusedextensivelyfortestingC.difficile

treatmentefficacy(Freemanetal.2007;Bainesetal.2008)andwillenablevarious

questions around the CD27L endolysin to be asked and answered, such as: how

quicklydoestheendolysindegrade/denatureinthehumangutenvironment?How

muchendolysinisrequiredtoachieveareductioninC.difficilebacterialloadandhow

oftenmustdosesbegiven?Whatdeliverymechanismislikelytoenablethedelivery

of CD27L to the key C. difficile infection sites? How does CD27L compare to

vancomycintreatment?CanCD27Lactsynergisticallywithvancomycintreatment?

However,majorissueswithevenanadvancedinvitromodelstillexist,suchasthe

absenceofimmunologicalinfluence.Toaddressthis,eventuallysomeformofinvivo

modelwill doubtlessbe required,but theuseof advanced in vitro testing should

reducetherelianceupontheseand informmorepreciselywhichtestsneedtobe

carriedout.

An interesting final considerationofC. reinhardtii-producedCD27L is thedelivery

mechanism. To date, C. reinhardtii-produced therapeutics have been considered

mostviablefortopicalapplicationsduetothepotentialcostsavingsmadebyusinga

crude extract, on the assumption that a crude extract would not achieve FDA

approval for systemic use. However, C. difficile has recently emerged as an

increasinglyprevalentpathogeninfoodanimals(Songer2004;Keeletal.2007),such

aspigs,calvesandostrichesaswellaspetssuchascats,dogsandhorses(Gouldand

Limbago2010).Theseofferapotentialmarketthatmaybelesstightlyregulatedand

enableC.reinhardtiiproducedtherapeuticstoshowaproof-of-concept.WorkbyDr

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PriscillaRajakumarinthePurtonLabinvestigatedtheadministrationoforalvaccines

produced in C. reinhardtii to chickens and fish, and discussed the use of the C.

reinhardtiicellasadeliverycapsule(Rajakumar2016).Whilethisconcepthasnot

been practically tested, Rajakumar (2016) suggests that the cell wall and the

chloroplast of C. reinhardtii protect the therapeutic recombinant proteins from

degradationthroughtheupperlevelsofthedigestivesystem,enablingthemtoreach

the gut. This will, of course, depend upon which animal is being treated, as for

examplethechickensstudiedbyRajakumar(2016)haveaveryshortintestinaltract,

whilethesamestrategymaynotworkincattle.

5.4.2 Gp20endolysin

TheGp20endolysinhasbeenproblematic since recombinantexpressionwas first

attempted by Taunt (2013). As an endolysin targeting a topical and prevalent

bacterialspecies,itwouldbeanidealcandidateforproductioninC.reinhardtiiasa

treatmentforacnevulgaris.However,previousattemptstoachievegp20expression

intheC.reinhardtiichloroplast,aswellasnewattemptsdescribedinthischapter,

haveallfailedtoproducedetectablelevelsofexpression.TotestwhethertheGp20

protein is stable and active outside the P. acnes intracellular environment, the

endolysinwasexpressed inE.coli. InE.coliGp20accumulated tohigh levels,but

colonyformingunitassaysusingacrudelysateagainstP.acnesfailedtoshowany

activity.GivenhighlevelsofinterestinP.acnes,andtheidentificationofatleast14

relevant putative endolysins, it does seem remarkable that no reports of a

recombinantly produced endolysin targeting P. acnes exist, other than the

aforementionedpatentapplicationbyLysando.Herewewilldiscussinmoredetail

thepossiblereasonsforissuesfacedinexpressionandactivity,andpotentialfuture

linesofinvestigation.

Afterfailedinitialattemptstoexpressgp20 intheC.reinhardtiichloroplast,Taunt

(2013) investigated whether translation initiation was the issue by creating a

cpl1:gp20 fusion, but again expression failed, while a positive control cpl1:pal

succeeded.Thissuggestedthattheribosomewasstallingatsomepointwithinthe

gp20gene,anissuemostcommonlyassociatedwithcodonusage.However,further

233

codon optimisation failed to solve the problem. Taunt (2013)was also unable to

detectgp20expressioninE.coli,leadingtoasuggestionthatrapidproteinturnover

maybetoblame.However,stronggp20expressioninE.coliwasdescribedearlierin

this chapter,making this a less likely suspect, althoughadmittedly theE. coli and

chloroplastenvironmentsarenotidentical.

Asmentionedearlier,theremarkablesimilaritybetweenthe14P.acnes targeting

endolysinsdescribedsofarmeansthattestingtheexpressionofadifferentendolysin

isunlikelytomakeanydifference.ThepotentialdemandforsolutionstoP.acnes

infection from both the cosmetic and pharmaceutical industries means that this

endolysinremainsworthyofinvestigation.

ThealignmentperformedbyMarinellietal. (2012) (Figure5.17)providesauseful

indicationofthetwoendolysindomains,astheareaofleasthomologyislikelytobe

the glycine rich linker domain (circa position 200). This is further supported by

databasecomparisonspredictingthattheN-terminaldomainisassociatedwithN-

acetylmuramoyl-1-alanineamidaseactivity(Farraretal.2007).Giventhatendolysin

modulesgenerallyremainstablewhenseparated(Mayeretal.2008;Dunneetal.

2016),itwouldbeinterestingtoattempttoexpressthetwoseparatedomainsinthe

C. reinhardtii chloroplast. This would give an insight into where the ribosome is

stalling,andcouldpotentiallybesubjectedtofurthercodonoptimisationtominimise

this.Furthermore,asdemonstratedwithCD27L,occasionallytheendolysincatalytic

domainaloneissufficientforcelllysiswhichcouldbetestedhereifexpressioncan

beachieved.

234

FigureRemoved

Figure5.17Proteinsequencealignmentof14putativeendolysinsfromP.acnesbacteriophages.Thealignmentshowsthatthesequenceishighlyconserved,withthemajorityofdifferenceslocatedintheglycine-richlinkerataroundposition200.TheC-terminuscellwallbindingmoduleislesshighlyconservedthanthecatalyticN-terminusmodule.Gp20fromthePA6bacteriophageisthe4thfromthebottom,labelledPA6_rev_gp20,asitisarevisedannotationofthePA6genome.ReproducedfromMarinellietal.(2012).

235

ThenextissuetoresolveistheapparentlackofactivityoftheE.coli-producedGp20

proteinagainstP.acnes.ThefirstquestiontoaddressinthiscaseistheCFUassay

itself,whichwasnotfullysatisfyingandwasnotrepeatedduetotimeconstraints.

The investigation requiresa suitablepositivecontrol, theobviouschoicebeingan

antibiotic such as erythromycin, the first line of treatment in a clinical setting

(Zaenglein et al. 2016). However, it would be preferable to have amore specific

antibacterial,whichwillpartiallyconfirmthatthebacteriaseenareindeedP.acnes

andnotacontaminatingspecies.Thepeptideadrenomedullinissecretedbyhuman

epithelial cells and has been demonstrated to have an antimicrobial effect upon

severalmicroorganisms foundontheskinandrespiratory tract,but isparticularly

effectiveagainstP.acnes,M.luteus,P.gingivalisandH.pylori(Allakeretal.1999).It

thereforerepresentsanarrower-spectrumantimicrobial,whenusedatanoptimal

concentration, than erythromycin and may provide a useful positive control. To

furtherconfirmthatthebacteriuminquestionisP.acnes,Gramstain(+),catalase(+)

andindole(+)tests,followedbysequencingofthe16SrRNAgeneforspecies-level

identificationcanbeperformed(Butler-Wuetal.2011).

Assumingthattheaboveassayisimprovedtoanacceptablestandardbutactivityis

stillnotobserved,severalissuescouldbeatplay.ThefirstisthatP.acnesisknown

tosecretemanyprotectiveenzymeswhichallowittosurviveintheharshhumanskin

environment, includinghydrolases andproteases (Holland et al. 2010), aswell as

propionic and acetic acid which inhibit the growth of competing species (Wilson

2005).Itisplausiblethatsomecombinationofthesefactorsiscausingtheendolysin

todenatureorbedegradedbeforeitcanbindandlysethecellwall.Astheendolysins

have evolved to be active from within the bacterial cell, it is possible that such

extrinsicbarriersareblockingitsaction.Anotherpossibilityisthattheholin,whichis

normallyexpressedalongsidetheendolysin, isrequiredforcell lysis,althoughthis

seemsunlikelyandhasnotbeenreportedbefore,totheauthor’sknowledge.

Asseenearlier in thischapter,whilemostendolysinsconsistofa simplemodular

monomer,otherssuchasPlyCaremultimeric,andstillotherssuchasCTP1Lrequire

oligomerisationwiththeirowntruncatedC-terminaldomain.Itispossiblethatthe

lackof activity observedwithGp20 is due to the requirement tooligomerise like

236

CTP1L for full activity. Figure 5.17 shows a conserved methionine residue at

approximatelyposition193whichcouldbeaninternalstartcodon.Alsoatposition

210inFigure5.17thealternativestartcodon,GTG,codesforavaline.Thisresidueis

conserved as either a methionine or a valine in the other putative endolysins,

howeverananalysisofthenucleotidesequenceisrequiredtoidentifywhetherthis

isapotentialinternalstartcodon.ThislocationissimilartothatobservedinCTP1L

andCD27Landwouldsimilarly result in thesynthesisofa freeCBD.However,no

ShineDalgarnoconsensuscanbeseenupstreamofeitherthemethionine-encoding

ATGorvaline-encodingGTGwhenthenucleotidesequenceofgp20isexamined,and

analysis using the NCBI Open Reading Frame Finder

(https://www.ncbi.nlm.nih.gov/orffinder/) software did not identify either as a

potentialORF(Figure5.18).

237

Figure5.18Resultsfromhttps://www.ncbi.nlm.nih.gov/orffinder/withtheputativeendolysinsequencegeneID5247034.OnlyORF1isinframeandinthe

samedirectionasthefull-lengthendolysin.ORF1is654nucleotidesinlength,producinga217aminoacidprotein.

238

TheinternalribosomebindingsiteandORFidentifiedinFigure5.18wouldresultin

thesynthesisofa freeCBD, likeCTP1L,but includesamuch largerportionof the

catalyticdomainthanwouldbeexpected.Nevertheless, itwouldbe interestingto

attempttoexpressgp20 inE.coliusingthewild-typenucleotidesequence,which

mayidentifywhetheraninternalribosomebindingsiteisactive.Suchafindingmay

resultintheimprovementofactivityagainstP.acnes.

5.4.3 Futureendolysins

Asdiscussedearlierinthischapter,theproblemssurroundingGp20cannotbesolved

bymerelyattemptingtoexpressanalternativeP.acnestargetingendolysin,asthe

levelofhomologybetweentheproteinsissohighthatitisextremelyunlikelythat

this will make any difference. In this case the strategy needs to be to better

understand why the endolysin is failing to express and attempting to solve any

problemswithactivity.

ThereareseveralotherendolysinswhichwouldbeinterestingtoexpressintheC.

reinhardtii chloroplast in the future. CTP1L is an interesting endolysin due to its

requirement tooligomerisewith its freeCBD,but is also a commercially relevant

endolysin, as it targets Clostridium tyrobutyricum. Clostridium tyrobutyricum

contaminationcanleadtoextensivecostsinthecheeseproductionindustrythrough

‘late-blowing’, a phenomenon affecting high-pH cheeses such as Edammer and

Gouda,andresultinginexcessbubblesandanabnormalflavour.Anapplicationof

CTP1L istherefore infoodsafetyandproduction,andgiventhatC.reinhardtiihas

GRASstatus,thismaywellbeaverywellsuitedmarketforC.reinhardtii-produced

endolysins.

WorkbyJadoetal.(2003)demonstratedthattwoS.pneumoniaeendolysins,Paland

Cpl-1,areabletoactsynergistically.Therearenumerousadvantagestoexploiting

drug synergies therapeutically, such as lower dosages, greater efficacy, and

minimising the opportunity for resistance to occur. It would be interesting to

investigate whether a similar synergy can be observed with endolysins targeting

otherbacteria,suchasC.difficile.Wangetal. (2015) identifiedanotherC.difficile

endolysin,namedPlyCD,whichwasdemonstratedtobeactiveagainstC.difficilein

239

vitroand,similarlytoCD27L,moreactiveinitstruncatedform,PlyCD1-174.Successful

expressionofPlyCDintheC.reinhardtiichloroplastwouldallowtestingofbothPlyCD

as a stand-alone endolysin, as well as enabling an investigation into whether a

synergisticeffectcanbeobservedbetweenPlyCDandCD27L.

As a final note on selecting future endolysins, one method might be termed a

‘disease-orientated’ approach. This approach looks at the bacterial species

associatedwithcertainidentifiablediseasesanddesigning‘endolysincocktails’tokill

allofthosespecies.Forexample,theskindiseaseimpetigoistypicallytreatedwitha

broad-spectrumantibioticcream(GeorgeandRubin2003),despitethefactthatitis

knowntobecausedbyoneofonlytwobacterialspecies:Staphylococcusaureusor

Streptococcuspyogenes. It is thereforeconceivablethatacocktailof twoormore

endolysins targetingS. aureus andS. pyogeneswouldbe a viable treatment. This

particular example, as a topical application, would again be well suited to C.

reinhardtii production. Two potential endolysins, in this case, could be LysH5

(targetingS.aureus)(Gutiérrezetal.2014)andPlyC(targetingS.pyogenes)(Shenet

al.2013).

Overall,thischapterhasdemonstratedthatwhiletheC.reinhardtiichloroplastcan

beaverywellsuitedplatformforendolysinproduction,thereremainseveralissues

and unexplored areas which leave recombinant endolysin expression far from

routine.However,non-expressingendolysingenessuchasgp20offeranopportunity

tounderstandmoreabout factorsaffectingchloroplastexpression, suchascodon

optimisationandtranslationelongation.Thesuggestedfutureworklaidoutinthis

section may help us to progress the C. reinhardtii chloroplast platform towards

achievingmorereliableproteinproduction.

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Chapter6 Finaldiscussionandfutureprospects

6.1 Summary

6.1.1 Summaryofresults

Theresearchdescribedinthisthesissoughttoaddresstwooverarchingissues.The

first is the improvement of the genetic engineering technology underpinning the

developmentof theC. reinhardtiichloroplastasanovelplatformforrecombinant

proteinproduction.Thesecondistheadvancementofendolysinsasanalternative

toconventionalantibiotics.

Chapter3investigatestheissueofrelativelylowlevelsoftransgeneexpressionseen

inC.reinhardtiianddemonstratestechniquesavailabletoimprovethis.Asdemand

grows for protein therapeutics, such as monoclonal antibodies, hormones (e.g.

humaninsulin),andvaccines,sodoestherequirementforsuitableplatformsfortheir

recombinantproduction.Asdiscussedin1.3.1,therearevariousplatformscurrently

used for therapeutic protein production, each with different advantages and

limitations.ThegreenalgaC.reinhardtiioffersanovelalternativecellfactory,with

importantadvantagesover severalmorewidelyusedplatforms.However, several

limitationsofC.reinhardtii,includinglowlevelsofexpression,needtobeaddressed

beforeitcanrealisticallycompetewiththeestablishedplatforms.Initially,methods

used to compare protein accumulation levels between different transgenic C.

reinhardtii lines were investigated. The chapter highlights the limitations of

comparingproteinaccumulationlevelswhilstclonalvariationappearstobehaving

such a large effect, as well as proposing and testing improved methods of

comparison.Assuch,attemptstoinvestigatetheeffectsofcodonpairoptimisation

remaininconclusive.However,thechapterdoesdemonstratethattheintroduction

oftwoexpressioncassettesintotheC.reinhardtiichloroplastplastomeresultsina

robusttwo-foldincreaseinproteinaccumulationcomparedwithasingleexpression

cassette.

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Thegrowingthreatofantibioticresistancehasencouragedresearchintodeveloping

alternativestoconventionalantibiotics.Endolysinsareofincreasinginterestandthe

first endolysin based therapies arenowavailable (1.3.3.3). Chapter 4 investigates

whether the activity of these endolysins can be improved against their target

bacterium, S. pneumoniae, either by direct mutation of the protein or through

synergisticeffectswithholins.Sitedirectedmutagenesiswasusedtodisruptthecell

wallbindingdomainofCpl-1andexpressionwasachievedforallsixmutants inC.

reinhardtii.However,suchmodificationsappearedonlytodecreasetheactivityof

theendolysin,demonstratingthatthefunctionalityofthetwoactivecholinebinding

sitesisessentialfortheactivityoftheendolysininthisinstance.DimerisationofCpl-

1viaadisulphidebridgewasalsoinvestigated,butcouldnotbeachievedintheC.

reinhardtii chloroplast. Several reasons for the failure of Cpl-1 to dimerise were

suggested, along with possible solutions, mostly revolving around the steric

hindrance of the C-terminal epitope tag. Finally, the holin protein Cph-1 was

expressedintheC.reinhardtiichloroplast.However,expressionquicklyceasedand

thepossiblereasonsforthiswerediscussed.

Chapter 5 describes attempts at increasing the suite of endolysins that can be

producedintheC.reinhardtiichloroplast.TheC.difficiletargetingendolysin,CD27L,

wassuccessfullyexpressedintheC.reinhardtiichloroplastanditsactivityagainstC.

difficile was demonstrated in vitro. Meanwhile, the P. acnes targeting endolysin,

Gp20, failed to express in transformed C. reinhardtii lines. However, it was

successfullyexpressedinE.coliunderaninduciblepromoter,butactivitycouldnot

beobservedagainstP.acnes.

6.1.2 Summaryofshort-termfuturework

Futureworkhasbeenroughlydividedintotwocategoriesinthisthesis:‘short-term’

futureworkand‘long-term’futurework.Whilebothofthesetermsarerelative,the

goalofthissystemistodistinguishbetweenworkthatwillenabletheprogressionor

conclusionofcurrentprojects(short-term),andworkthatlooksfurthertothefuture

andwouldserveasastand-aloneprojectinitsownright(long-term).Relevantshort-

242

termfutureworkhasbeensuggestedattheendofeachresultschapter,muchof

whichisveryspecific.However,themostinterestingareathateachresultschapter

highlightedforfurtherinvestigationissummarisedbrieflybelow.Potentialpathsfor

long-termfutureprojectsarediscussedin6.2below.

TheissueofclonalvariationhighlightedinChapter3limitsourabilitytoaccurately

investigate strategies for improving recombinant protein accumulation in the C.

reinhardtiichloroplast,butalsooffersanopportunitytounderstandwhatleadstoa

veryhighor very lowexpressing ‘clone’. Inorder toenable the investigation into

subtlermethodsofimprovingrecombinantproteinaccumulation,in3.4.2wesuggest

thattheuseofareportergene,suchasluciferase,andtheuseofhigh-throughput

techniques will enable ‘comparison-of-means’ analysis. Advancements in whole

genome sequencing, and importantly its falling cost, may eventually enable a

methodicalwholegenomeanalysisofhigh-expressingclonestoidentifymutationsor

insertionsthatareresultinginhighlevelsofproteinaccumulation.Thesemutations

might be affecting nuclear genes for chloroplast regulatory factors, proteases, or

chaperones. These changes can thenbe purposefully introduced alongside future

transgenes,thusimprovingC.reinhardtiiasaproteinproductionplatform.

TheendolysinCpl-1wasthemainfocusof investigation inChapter4,andassuch

much of the proposed future work was specific to this endolysin. However, the

unexpected andunexplained result thatCph-1 expressionwas achieved and then

ceased isworthyof further investigation.Silencingmechanisms,whilecommon in

nuclear transformations, have not previously been described in the chloroplast

(Danielletal.2005).Itisthereforeimportanttounderstandhowtheterminationof

expression has occurred, the findings of which may help prevent this from

reoccurring,butalsocouldbecomeausefulmolecularswitch.

Chapter5demonstratedthesuccessfulexpressionandactivityofasyntheticgene

encodingCD27L,andalsodocumentedtheunsuccessfulattemptstoexpressgp20in

C. reinhardtii. However, it is thegp20 result that remains interesting in terms of

gainingagreaterunderstandingofC. reinhardtiiexpressionand itssuitabilityasa

platformforproducingendolysins.Despiterepeatedattemptsandfailurestoachieve

243

gp20expressionintheC.reinhardtiichloroplast,thereisstillsomewaytogobefore

we can deduce the precise reason for this and attempt to ameliorate it. Codon

optimisation, translation initiation, and epitope tag location have all been

investigatedanddonotappear tobecausing failureofexpression.Attempting to

expressthetwodomainsoftheendolysinseparatelymayshedsomelightontothe

problem, and aid us in a broader understanding of expression failure in the C.

reinhardtiichloroplast.

6.2 Discussionandlong-termfutureprospects

6.2.1 AlternativestrategiestoimprovingexpressionlevelsinC.reinhardtii

chloroplast

6.2.1.1 Syntheticpromotersand5’UTRs

Syntheticpromotersand5’UTRsarebecomingwidelyusedinsyntheticbiologyand

in recombinant protein production, due to their frequent ability to providemore

consistent and higher final protein yields than endogenous regulatory elements

(Gilman and Love 2016). As discussedpreviously in this thesis (3.1.2), chloroplast

gene expression appears to bemostly controlled at the level of translation in C.

reinhardtii,ratherthantranscription,andassuchtheimprovementof5’UTRsisof

greater interestthanthatofpromotersatthisstage.Todate,themostconsistent

levelsofgeneexpressionintheC.reinhardtiichloroplasthavebeenachievedusing

endogenouspromoter/5’UTRsequencesfromhighlyexpressedproteins,andinsome

caseshigherlevelshavebeenachievedthroughcombinationswithnuclearmutants

(Micheletetal.2011)andalteredCESmechanisms(Manuelletal.2007)whichwill

bediscussedisthenextsection(6.2.1.2).

ResearchbySpecht&Mayfield(2013)useda luciferasereportergene(Figure6.1)

andhigh-throughputanalysistocompileanunbiaseddatasetofmutatedpsaAand

psbD5’UTRs,fromwhichthefirstsuccessfulsyntheticC.reinhardtiichloroplast5’UTR

was designed. The resulting synthetic 5’UTR improved protein accumulation by

approximately66%,comparedtoendogenous5’UTRs.

244

FigureRemoved

Figure6.1C.reinhardtiitransformedwithexpressioncassettescontainingmutated5’UTRscontrolling luciferase expression in the chloroplast. This false colour image illustrates thedifferencesinluciferaseexpressionunderdifferent5’UTRs.TechniquessuchasthisenabletheprocessingandexpressionquantificationofhundredsofC.reinhardtiicoloniesinaverysmall amount of time. The positive control (wild type UTR) and negative control (no-luciferase)areindicatedonthebottomleftoftheplate.ReproducedfromSpecht&Mayfield(2013).

Ashigh-throughputtechniquesbecomemoreaccessibleanddefinedinC.reinhardtii,

thepotentialforproducinglargeunbiaseddatasetsimproves.Theavailabilityofsuch

datasetsmayenabletheidentificationofconsensusmotifsinboththepromoterand

5’UTRs of endogenous genes and pave the way towards producing synthetic

promotersand5’UTRsthatdramaticallyimproveproteinyieldsaswellasproviding

consistentlevelsofgeneexpression.TheworkbySpecht&Mayfield(2013)isthefirst

step in achieving this, but their use of particle bombardment to transform C.

reinhardtiirecipientlinesremainssubjecttotheissueofclonalvariation,andassuch

veryprecisedeductionsabout5’UTRconsensussequencesaredifficulttodrawand

anevenlargerdatasetislikelytoberequired.Recentadvancementsinprecisiongene

editingmayprovidethesolutiontothisissue,aswillbediscussedinthefollowing

section.

6.2.1.2 ThepotentialofCRISPR/Cas9geneediting

Asanalternativetomodifyingthe5’UTRwhichisregulatedbyvariouselementsin

the C. reinhardtii chloroplast (a cis-acting element), one could also modify the

245

regulatory elements themselves (trans-acting factors). Such an experiment was

performedbyMicheletetal.(2011),wherechloroplasttransgeneswereshowntobe

upregulated when expressed in a cellular background that included a nuclear

mutation.Themutationinthisinstancewaswithinatrans-splicingfactorthatwas

importedtothechloroplastfromthenucleus,thuspreventingtrans-splicinginthe

chloroplast.However,thismutationalsoledtoalossofphotoautotrophymeaning

thattheC.reinhardtiicellsmustbegrowninthedarkandrequireacetateasacarbon

source.ThediscoveryofCRISPR/Cas9geneediting,andtherecentapplicationofthis

technologytotheC.reinhardtiinucleus(Baeketal.2016;Shinetal.2016),willresult

inexperimentssuchasthatdescribedbyMicheletetal.(2011)becomingfareasier

to perform since targeted knock-outs of nuclear geneswouldbepossible. Rather

thanknockingouttrans-actingelementssuchassplicingfactorswhich,asdescribed,

result in other undesirable phenotypes, CRISPR/Cas9 could be used to knock-out

other factors that indirectly control protein accumulation, such as chloroplast-

targetedproteases.Theeasewithwhichsuchmutationscouldbeintroducedshould

quicklyenableresearcherstomanipulatethechloroplastenvironmentintoonethat

is more amenable to recombinant protein accumulation without having large

detrimentaleffectsoncellphysiology.

6.2.1.3 Expressionofnovelchaperones

As discussed earlier, the primary suspect for causing low rates of successful

recombinantproteinexpressionintheC.reinhardtiichloroplasthasbeentranslation

initiationandelongation.However,poorproteinstabilityorhighproteolysiscould

alsobeplayingarole inthis.Misfoldedproteinsandthosethathavenotreached

theirnativestatearetargetsofproteases.Whileknockingoutproteasesmaybepart

of the solution to improving protein accumulation, the quality of protein being

produced is equally important, and disruption of proteases may have other

detrimentaleffectsuponthecell(RamundoandRochaix2014).

WhilerecombinantproteinyieldsinE.colihavebeensteadilyimprovedoverthepast

four decades, through a process of largely trial and error, we must attempt to

reproduce these advances inC. reinhardtiiby analysing those improvements and

246

specificallyintroducing.ManyofthesuccessfulimprovementsinE.coliweredueto

theco-expressionofchaperones(Makinoetal.2011),andthereforethisisworthyof

investigation. Many chaperones are known to be expressed in the C. reinhardtii

nucleus and targeted to the chloroplast to aid protein folding (Schroda 2004).

However,somenovelchaperonesmayhavespecificabilitiestoaidinthefoldingof

certain recombinant proteins, and studies in E. coli have found that co-

overproductionofcertainchaperonesintheE.colicytoplasmhaveaidedinprotein

solubility and yields (deMarco et al. 2007). Itwould, therefore, be of interest to

identifyrelevantchaperones,suchasHeatShockProtein70(Willmundetal.2008),

andattempttoexpressthemalongsideendolysins intheC.reinhardtiichloroplast

withthegoalofimprovingproteinaccumulationandexpressionsuccessrates.

6.2.2 Othermicroalgalhostsforscale-up

C.reinhardtiihasbeenanimportantmodelorganismforthestudyofphotosynthesis,

chloroplastbiogenesis,andflagellarfunctionfordecades(Rochaix1995).Giventhe

years of research behind it and its well-defined genetics, C. reinhardtii was the

obvious initial candidate for proving the concept ofmicroalgae as a recombinant

protein production platform. However, when it comes to scaling up microalgae

production for commercial exploitation, other speciesmaybemore suitable, and

researchperformedonC.reinhardtiishouldbereadilytransferabletothesespecies.

Forexample,somespeciessuchasthoseofSpirulina,Chlorella,Haematococcus,and

Dunaliellahaveallbeengrownoncommercialscalesforsometime(Figure6.2)and

arewellsuitedtocultivation(Rosenbergetal.2008).

247

FigureRemoved

Figure 6.2 Spirulina growing in open ponds (raceways) for commercial production atCyanotech’sfacilitiesinHawaii2.

Theadvantagetousingoneoftheabovemicroalgaeisthatthetechnologyalready

exists fortheir largescaleproduction,aswellasdataongrowthoptimisationand

commercialviability.WhileyieldsoftherapeuticproteinsfromC.reinhardtiiarevery

low, hovering around 5-8mg L-1culture volume (1.3.3.3), the potential for cheap

cultivationandhighvolumesgoessomewaytowardsnegatingthisissue.Incontrast

CHO cells, as an example, must be grown in highly controlled environments in

extremelyexpensivemedia,itisunderstandabletheiryieldperlitremustbeveryhigh

tomakethiseconomicallyviable.Itisthereforeplausibletoexpectthatmovingtoa

newalgalplatformcouldbeeconomicallyfeasiblewithoutrequiringvastincreasesin

proteinyield.

Othermicroalgaeofinterestincludeextremophiles,becausetheircultureconditions

generallyminimisecontamination,whichisabigissuewhengrowingmicroalgaeat

the commercial scales and open raceways shown in Figure 6.2. For example,

Cyanidioschyzonmerolaeisamicroalgathatinhabitshot(40–56°C)andacidic(pH

0.2-4)environments,anditschloroplasthasrecentlybeentransformedforthefirst

time(Zienkiewiczetal.2017).Attemptingtoexpressrecombinantproteins,suchas

Cpl-1,inC.merolaeandsimilarorganismswillbeanimportantstepindetermining

2https://www.cyanotech.com/company/facility.html

248

thebest option for commercial scale-up.However, useof high temperatures and

extremophiles will require investigation into the thermostability of the proteins

lookingtobeproduced,andwillnotbesuitableforallproducts.Furthermore,the

cost of maintaining cultures at such temperatures may negate or minimize the

savingsmadebyadoptingsuchasystem.

6.3 Concludingremarks

6.3.1 Viewsonthefutureofantibiotics

As with many emerging technologies, the adoption of endolysins as antibiotics

dependsheavilyuponthestatusoftheexistingtechnology:conventionalantibiotics.

Therecentdiscoveryofanewantibiotic,teixobactin,andthenovelmannerinwhich

it was discovered (see 1.1.2.2) may yet extend the ruling era of small molecule

antibiotics.Whilethediscoveryofnewantibioticsisclearlyagoodthing,itisvitally

importantthatcomplacencydoesnot limittheresearch intoendolysinsandother

antibiotic alternatives. Conventional antibiotics have clear disadvantages when

compared toendolysins–namely thehugeselectivepressure theyexertuponall

bacteria to form resistance, thus making us increasingly dependent upon new

discoveries. It is of great importance, therefore, that research continues into the

developmentofmoreadvancedantibioticssothatwecanspreadourrelianceacross

several strategies rather than continuing to depend upon the discovery of new

conventionalantibiotics.Withapproximately1031bacteriophageinexistenceonthe

planet,thereisavast,andconstantlyevolving,supplyofpotentialantibioticsinthe

formofphage-therapyandendolysins,offeringamuchmorestableandsustainable

sourceoffuturetherapies.

For full adoption of endolysins and replacement of conventional antibiotics, both

much improved point-of-care diagnostics, and much more research into tackling

Gram-negativebacteriaare required.Bothof theseare stumblingblocks thatwill

take time and resources to overcome, but the final result will be a far superior

solution to tackling bacterial infections. Furthermore, if endolysins, or any other

technology, do become the first-line treatment for bacterial infections, research

249

shouldconstantlystrivetofindalternatives,asthe‘arms-race’withbacteriaisnever-

ending,andresistancetoanynewtechnologieswilleventuallyemerge.

6.3.2 Viewsonthefutureofmicroalgae-producedtherapeutics

Hypothetical conversationswith drug regulatory bodies and thought-experiments

around the advantages of algae-based therapeutics are clearly important initial

stages of developing a new recombinant protein platform, and currently more

immediate issues such as low recombinant protein yields are preventing

advancementoftheplatform.However,toperformacostcomparisonofmicroalgae

and other platforms, and to establish the viability of the platform, it is vitally

important to understand the extent to which drug regulatory bodies will look

favourablyuponproteinsproducedinGRASorganismssuchasC.reinhardtii.Ifitis

the case that crude or enriched algal extracts can be approved, then thiswill be

enormouslyencouragingforthefutureoftheplatform.However, ifthis isnotthe

case, thentheuseofestablishedplatformswillalmostalwaysbemore financially

favourable. In this situation, the future of microalgae as recombinant protein

production platforms must shift towards alternative markets, such as cosmetics,

veterinarymedicine, and food safety, and look to other proteins that cannot be

expressedinothersystemsorhavemarkedsynergieswithalgalcrudeextracts.Other

proteinproductionplatformswithGRASstatus,suchasspinach(Schulzetal.2015),

andpossiblyeventhebacterialsystemLactococcuslactis(Cano-Garridoetal.2014),

arelikelytoreachthepointofcommercialviabilitysoonerthanmicroalgaeandthis

willoffervaluableinsightsintohowtheseproteinsarereceivedandregulatedbythe

relevantregulatorybodies.

250

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Chapter7 Appendices

7.1 ResultsChapter3Appendices

7.1.1 AppendixA

Figure 7.1 Total protein staining of five technical repeats of TN72_SR_cpl1-HA andTN72_SR_cpl1-HA_A_cpl1_c-HA. These data were used for normalisation of the anti-HAsignaltoaccountforloadingerror.

(kDa)

275

7.1.2 AppendixB

Figure7.2DemonstrationofhomoplasmicityofTN72_SR_cpl1-StrepII.PrimersusedinthisinstancewereFlank1,psa.R,RbcL.Fwhichprovidean850bpbandfortherecipientstrainanda1135bpbandforahomoplasmicstrain.

276

7.1.3 AppendixC

Figure7.3TotalproteinstainingusedtonormaliseforloadingerrorswhencomparingtotalproteinaccumulationbetweentheaboveC.reinhardtiilines.

(kDa)

277

FigureRemoved

7.1.4 AppendixD

Figure7.4CodonfrequencychartgeneratedforC.reinhardtiichloroplasttransgenecodonoptimization,fromCodonUsageOptimizerBeta0.92(Kong2013).

7.1.5 AppendixE

Figure 7.5 Western blot analysis demonstrating the varying levels of HA-tagged proteinaccumulationovera194hourcultureperiod.

0 unidentified codons.Nucleotide frequency: A=29 U=45 G=15 C=10 X=0 GC Content: 25.25% 1st letter GC: 36.36% 2nd letter GC: 27.27% 3rd letter GC: 12.12% Summary table:Sequence length: 21279 Number of protein genes: 32 Total codons: 7093

0 unidentified codons.Nucleotide frequency: A=5880 U=7318 G=4106 C=3975 X=0 GC Content: 37.98% 1st letter GC: 50.53% 2nd letter GC: 41.41% 3rd letter GC: 21.99% Weight table from reference set:

CodonUsageOptimizerBeta 0.6

Amino acid | Codon | Number | Frequency per thousandPhe UUU 1 30.3 Ser UCU 0 0.0 Tyr UAU 1 30.3 Cys UGU 0 0.0

UUC 0 0.0 UCC 0 0.0 UAC 1 30.3 UGC 0 0.0Leu UUA 6 181.8 UCA 0 0.0 STOP UAA 1 30.3 STOP UGA 0 0.0

UUG 0 0.0 UCG 0 0.0 UAG 0 0.0 Trp UGG 0 0.0Leu CUU 3 90.9 Pro CCU 0 0.0 His CAU 0 0.0 Arg CGU 0 0.0

CUC 0 0.0 CCC 0 0.0 CAC 0 0.0 CGC 0 0.0CUA 0 0.0 CCA 0 0.0 Gln CAA 0 0.0 CGA 0 0.0CUG 0 0.0 CCG 0 0.0 CAG 0 0.0 CGG 0 0.0

ile AUU 2 60.6 Thr ACU 1 30.3 Asn AAU 0 0.0 Ser AGU 1 30.3AUC 2 60.6 ACC 0 0.0 AAC 0 0.0 AGC 0 0.0AUA 0 0.0 ACA 2 60.6 Lys AAA 2 60.6 Arg AGA 0 0.0

Met AUG 1 30.3 ACG 0 0.0 AAG 0 0.0 AGG 0 0.0Val GUU 2 60.6 Ala GCU 1 30.3 Asp GAU 0 0.0 Gly GGU 4 121.2

GUC 0 0.0 GCC 0 0.0 GAC 0 0.0 GGC 0 0.0GUA 2 60.6 GCA 0 0.0 Glu GAA 0 0.0 GGA 0 0.0GUG 0 0.0 GCG 0 0.0 GAG 0 0.0 GGG 0 0.0

Amino acid | Codon | Number | Frequency per thousandPhe UUU 171 24.1 Ser UCU 147 20.7 Tyr UAU 87 12.3 Cys UGU 61 8.6

UUC 285 40.2 UCC 0 0.0 UAC 152 21.4 UGC 3 0.4Leu UUA 544 76.7 UCA 144 20.3 STOP UAA 30 4.2 STOP UGA 0 0.0

UUG 5 0.7 UCG 9 1.3 UAG 2 0.3 Trp UGG 170 24.0Leu CUU 152 21.4 Pro CCU 119 16.8 His CAU 23 3.2 Arg CGU 245 34.5

CUC 0 0.0 CCC 2 0.3 CAC 148 20.9 CGC 10 1.4CUA 53 7.5 CCA 200 28.2 Gln CAA 218 30.7 CGA 2 0.3CUG 9 1.3 CCG 13 1.8 CAG 10 1.4 CGG 0 0.0

ile AUU 343 48.4 Thr ACU 210 29.6 Asn AAU 83 11.7 Ser AGU 80 11.3AUC 102 14.4 ACC 3 0.4 AAC 198 27.9 AGC 30 4.2AUA 4 0.6 ACA 193 27.2 Lys AAA 240 33.8 Arg AGA 8 1.1

Met AUG 182 25.7 ACG 12 1.7 AAG 9 1.3 AGG 2 0.3Val GUU 258 36.4 Ala GCU 433 61.0 Asp GAU 135 19.0 Gly GGU 586 82.6

GUC 0 0.0 GCC 9 1.3 GAC 115 16.2 GGC 24 3.4GUA 261 36.8 GCA 173 24.4 Glu GAA 311 43.8 GGA 19 2.7GUG 6 0.8 GCG 23 3.2 GAG 20 2.8 GGG 7 1.0

TN72_SR_cpl1-HA

45h

TN72_SR_CPOCv1-HA

TN72_SR_Control

TN72_SR_CPOCv2-HA

53.5h

66.5h

97h

122h

139h

194h

278

7.1.6 AppendixF

Aone-wayANOVAtestwascarriedoutateachtimepointe.g. t=45 forsamples

taken at 45 hours into the experiment. In this experiment, four time timeswere

analysed,with3replicatesofeachonthewesternblot(Figure3.37).

Resultsatt=45Summary of Data

TreatmentsCPOCv1 CPOCv2 cpl1-HA Total

N 3 3 3 9∑X 0.115 0.4351 0.1253 0.6754Mean 0.0383 0.145 0.0418 0.075∑X2 0.0044 0.0632 0.0054 0.073Std.Dev. 0.0035 0.0066 0.0075 0.0528Result DetailsSource SS df MS

Between-treatments 0.0221 2 0.011

F = 293.57558

Within-treatments 0.0002 6 0Total 0.0223 8The f-ratio value is 293.57558. The p-value is < .00001. The result is significant at p < .05.

Results at t= 58Summary of Data

TreatmentsCPOCv1 CPOCv2 cpl1-HA Total

N 3 3 3 9∑X 0.3091 1.5043 0.2813 2.0948Mean 0.103 0.5014 0.0938 0.2328∑X2 0.0319 0.7607 0.0274 0.82Std.Dev. 0.0038 0.0565 0.0221 0.2038Result DetailsSource SS df MS

Between-treatments 0.325 2 0.1625

F = 131.77838

Within-treatments 0.0074 6 0.0012Total 0.3324 8The f-ratio value is 131.77838. The p-value is .000011. The result is significant at p < .05.

279

Results at t= 77Summary of Data

Treatments1 CPOCv1 CPOCv2 cpl1-HA Total

N 3 3 3 9∑X 0.9648 1.4821 0.663 3.1099Mean 0.3216 0.494 0.221 0.3455∑X2 0.3137 0.8476 0.1472 1.3085Std.Dev. 0.0411 0.2403 0.0178 0.171Result DetailsSource SS df MSBetween-treatments 0.1144 2 0.0572

F = 2.87208

Within-treatments 0.1195 6 0.0199Total 0.2339 8The f-ratio value is 2.87208. The p-value is .133348. The result is not significant at p < .05.


Treatments1 CPOCv1 CPOCv2 cpl1-HA Total


F = 0.36948

Within-treatments 0.0261 3 0.0087Total 0.0325 5The f-ratio value is 0.36948. The p-value is .718715. The result is not significant at p < .05.

280

7.1.7 AppendixG

Aone-wayANOVAtestwascarriedoutateachtimepointe.g. t=45 forsamples

takenat45hours into theexperiment. In this second runningof theexperiment,

seventimepointswereanalysedbutwithonlytworeplicatesofeach(Figure7.5).


TreatmentsCPOCv1 CPOCv2 Cpl-1 Total


F = 34.67033

Within-treatments 0.0004 3 0.0001Total 0.0105 5The f-ratio value is 34.67033. The p-value is .008445. The result is significant at p < .05.

Resultsatt=53.5Summary of Data



F = 61.0499

Within-treatments 0.0001 3 0Total 0.003 5

The f-ratio value is 61.0499. The p-value is .003714. The result is significant at p < .05.

281

Resultsatt=66.5Summary of Data



F = 5.09397

Within-treatments 0.002 3 0.0007Total 0.0089 5

The f-ratio value is 5.09397. The p-value is .108497. The result is not significant at p < .05.




F = 4.82318


The f-ratio value is 4.82318. The p-value is .11554. The result is not significant at p < .05.

282




F = 30.59972






F = 20.00184



283




F = 16.38062



284


7.2.1 AppendixH

Figure7.6cph1sequenceasorderedfromIntegratedDNATechnologies.Thestartcodonishighlightedingreen.TheintroducedTGAcodonsfortryptophanarehighlightedinorange.Thestopcodonsarehighlighted inred.TheHAtag ishighlighted inpurple.SapIandSphIrestrictionsitesareindicatedbybluearrows.

285

7.2.2 AppendixI

Figure7.7DemonstrationofhomoplasmicityofTN72_SR_cpl1_dimer-HA.Primersused inthis instancewere Flank1, psa.R, RbcL.Fwhichproduce an850bpband for the recipientstrainandan1135bpbandforahomoplasmicstrain.

286


7.3.1 AppendixJ

Figure7.8cd27lsequenceasorderedfromIntegratedDNATechnologies.Thestartcodonishighlighted ingreen.The stopcodonsarehighlighted in red.TheHA tag ishighlighted inpurple.SapIandSphIrestrictionsitesareindicatedbybluearrows.

287

7.3.2 AppendixK

Figure 7.9 Demonstration of homoplasmicity of TN72_SR_cd27l-HA. Primers used in thisinstancewereFlank1,psa.R,RbcL.Fwhichproducean850bpbandfortherecipientstrainandan1135bpbandforahomoplasmicstrain.Sixtransformantsweretested.

7.3.3 AppendixL

Figure7.10DemonstrationofhomoplasmicityofTN72_SR_cd27l-StrepII.PrimersusedinthisinstancewereFlank1,psa.R,RbcL.Fwhichproducean850bpbandfortherecipientstrainandan1135bpbandforahomoplasmicstrain.Twotransformantsweretested.

3Kb

1Kb0.5Kb

TN72

TN72_SR_

cd27l-H

A

3Kb

1Kb0.5Kb

TN72 TN

72_SR_

cd27l-S

trepII

288

7.3.4 AppendixM

Figure7.11gp20NsequenceasorderedfromIntegratedDNATechnologies.Thestartcodonishighlightedingreen.TheintroducedTGAcodonsfortryptophanarehighlightedinorange.Thestopcodonsarehighlighted inred.TheHAtag ishighlighted inpurple.SapIandSphIrestrictionsitesareindicatedbybluearrows.

TG GTA TGC TCT TCA ATG GCA TAC CCA TAT GAT

GTA CCT GAC TAT GCT GTT CGT TAT ATT CCA GCT

GCT CAT CAC TCA GCA GGT TCT AAC AAT CCT GTT

AAT CGT GTA GTA ATT CAT GCA ACA TGT CCA GAT

GTA GGA TTT CCT AGT GCT AGC CGT AAA GGT CGT

GCT GTT TCA ACT GCT AAT TAC TTC GCA TCA CCA

TCT AGT GGT GGT AGC GCT CAC TAT GTG TGT GAC

ATT GGA GAA ACA GTA CAA TGT TTA TCT GAA AGT

ACT ATT GGA TGG CAC GCA CCA CCA AAC CCT CAT

TCA TTA GGT ATT GAA ATT TGT GCT GAT GGT GGT

TCT CAT GCT AGC TTT CGT GTG CCT GGT CAC GCA

TAT ACA CGT GAA CAA TGA TTA GAT CCA CAA GTT

TGG CCA GCT GTT GAA CGT GCT GCA GTA TTA TGT

CGT CGT TTA TGT GAC AAA TAC AAT GTA CCT AAA

CGT AAA TTA TCA GCT GCT GAC TTA AAA GCA GGT

CGT CGT GGT GTT TGT GGT CAT GTT GAT GTA ACT

GAC GCT TGA CAC CAA AGC GAT CAT GAT GAT CCA

GGA CCA TGG TTT CCT TGG GAC AAA TTC ATG GCT

GTT GTA AAC GGT GGT AGC GGT GAC AGC GGA GAA

TTA ACA GTT GCA GAT GTA AAA GCT TTA CAC GAT

CAA ATT AAA CAA TTA TCA GCA CAA TTA ACT GGT

AGC GTG AAC AAA TTA CAT CAC GAT GTT GGT GTT

GTA CAA GTG CAA AAC GGT GAC TTA GGA AAA CGT

GTA GAC GCT TTA AGC TGG GTG AAA AAT CCA GTG

ACT GGA AAA TTA TGA CGT ACA AAA GAT GCT TTA

TGG TCT GTT TGG TAT TAT GTA TTA GAA TGT CGT

AGC CGT TTA GAT CGT TTA GAA AGC GCT GTA AAT

GAC TTA AAA AAA TAA TAA GCA TGC GTC AGT ttt

t

SapI

SphI

289

7.3.5 AppendixN

Figure7.12DemonstrationofhomoplasmicityofTN72_SR_gp20N-HA.PrimersusedinthisinstancewereFlank1,psa.R,RbcL.Fwhichproducean850bpbandfortherecipientstrainandan1135bpbandforahomoplasmicstrain.Twotransformantsweretested.

7.3.6 AppendixO:ListofPrimers

Primersinorderofappearanceinthisthesis.

NAME SEQUENCE NOTES

Flank1 GTCATTGCGAAAATACTGGTGC

Primers for general sequencing, PCR and

homoplasmicity testing

RbcL.F CGGATGTAACTCAATCGGTAG

Rbcl.R CAAACTTCACATGCAGCAGC

Psa.R CATGGATTTCTCCTTATAATAAC

atpA.R ACGTCCACAGGCGTCGT

123a GGAGGGAAGTAGGCAGTAGC

psbH.R GCAACAGGAACTTCTAAAGC

psaSEQ AACTATTTGTCTAATTTAATAACC

3Kb

1Kb

0.5Kb

TN72

TN72_SR_

gp20N-HA

290

cpl1.F GCTCTTCAATGGTTAAAAAAAATGATTTATTC

Primers for the site-directed mutageneis of the cell wall binding domain

cpl1.R GCATGCTTATTAAGCATAATCTGGAAC

W202A_SDM.F GTACAGCTAAACAAGATTCAAAAG

W202A_SDM.R CTTTTGAATCTTGTTTAGCTGTAC

W209A_SDM.F CAAAAGGTGCTTGGTTTC

W209A_SDM.R GAAACCAAGCACCTTTTG

W223A_SDM.F CCATATAATAAAGCTGAAAAAATTG

W223A_SDM.R CAATTTTTTCAGCTTTATTATATGG

W230A_SDM.F GTGGTGTTGCTTATTATTTCG

W230A_SDM.R CGAAATAATAAGCAACACCAC

Cpl1_BssHII.F CAGGCAATTTGCTTACACCTTT Primers to introduce a BssHII restriction site into Cpl-1 Cpl1_BssHII.R TTAAGATAAGCGCGCTACATCC

cpl1_C45S.F ACATATTTAAATCCATCATTATCAGCTC

Primers to remove and introduce cysteine residues to create Cpl1_dimer

cpl1_C45S.R GAGCTGATAATGATGGATTTAAATATGT

cpl1_D324C.F GTGAATTAGCATGTAATCCTTCATTT

cpl1_D324C.R AAATGAAGGATTACATGCTAATTCAC

cd27l.F TGGTATTGCTCTTCAATGAAAATTTGTATTACT Primers for the replacement of the HA tag with a StrepII tag on cd27l

cd27l_StrepII.R ACTGACGCATGCTTATTATTTTTCGAATTGTGGGTGAGACCAGTTAATGTTTTTGTTTAAAACACCTTCAAC

gp20_NcoI.F CGTATACCCATGGTGCGTTA Primers to introduce an NcoI restriction site to gp20 gp20.R GATTGCGGATCCTTATTAGGC

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