PROTEIN EXPRESSION AND PURIFICATION 7, 237–246 (1996)ARTICLE NO. 0034

Expression, Isolation, and Characterization of a SignalSequence-Appended Chimeric Precursor Protein

Naheed Kaderbhai and Mustak A. Kaderbhai1

Institute of Biological Sciences, The University of Wales, Aberystwyth, Dyfed SY23 3DD, United Kingdom

Received August 8, 1995, and in revised form November 17, 1995

(4). This endopeptidase processes the N-terminal signalThis report describes the properties and the func- sequences in the secretory as well as the cytoplasmic

tional utility of an unprocessed precursor protein and outer membrane precursor proteins (5,6). Se-overproduced in Escherichia coli. The precursor pro- quence comparisons of signal peptidases of Gram-posi-tein is from a fusion between DNA sequences coding tive and Gram-negative organisms indicate that thefor the alkaline phosphatase signal sequence and the enzyme is highly conserved across the prokaryoticfull-length of rat liver cytochrome b5. The intact pre- kingdom (7). The Escherichia coli enzyme is a mono-cursor protein accumulated in the membranes repre- meric polypeptide of about 37 kDa, coded for by thesented to over 5% of the total bacterial protein. A pro- lepB gene and found N-terminally anchored in the cyto-cedure involving disruption of the bacterial cells by plasmic membrane exposing the catalytically activesonication, isolation of the membranes by differential core toward the periplasmic space (8). A variety of incentrifugation, solubilization with a polar solvent, and vivo and in vitro studies has shown that signal pepti-ion-exchange chromatography provided milligram dase accomplishes the processing of structurally di-quantities of the undegraded precursor in a homoge-

verse signal sequences in precursor proteins with highneous and soluble form. The chimeric precursor pro-fidelity (9). Site-directed mutagenesis studies suggesttein displayed a characteristic b-type hemoproteinthat the E. coli signal peptidase is a serine proteasespectrum, identical to that of the native cytochrometype with the catalytic mechanism involving an un-b5. The properties of the precursor protein have beenusual serine–lysine dyad (10,11). However, the enzymeexamined by a range of biophysical and biochemicalis insensitive to inhibition by all the classic inhibitorsmethods. Molecular modeling suggests an amphi-of proteases (12) and the reaction mechanism of signalpathic structure in which a fully preserved solublepeptidase remains obscure because of lack of detailedcore of cytochrome b5 is terminally bonded by hy-structure. Moreover, determinations of kineticallydrophobic interactions between the amino-terminalmeaningful activities of signal peptidase are presentlysignal sequence and the carboxy-terminal membrane

anchoring hemoprotein sequence. The precursor sub- hampered by the lack of substrate-level quantities ofstrate was recognized and efficiently cleaved by signal suitable precursor protein. Natural precursor sub-peptidase. q 1996 Academic Press, Inc. strates from normally growing cells have proved inap-

propriate for this purpose because they are short-lived(T1/2õ 1 min) and are unstable (13). Although recombi-nant approaches have been successfully applied forOver the last decade there have been significant ad-overproduction of full-length precursor proteins (14–vances in our understanding of the protein secretory16), the accumulation of the latter in the form of inclu-pathways of both the prokaryotes and eukaryotes (1).sion bodies, despite efforts to renature them (17), hasAs protein secretion is an essential bioactivity of allprecluded their uses in studying the processing eventsliving cells, the prokaryotic secretory pathway has beenand for assaying the signal peptidase activity.proposed as a viable target for intervention with novel

We have been using a mammalian cytochrome b5 (b5-therapeutic or biocidal agents (2). An essential compo-T)2 as a model protein to investigate protein topogen-nent of the prokaryotic secretory pathway (3) identified

as a potential biocidal target is the signal peptidase2 Abbreviations used: b5, globular domain of cytochrome b5; b5-T,

full-length cytochrome b5; SS, signal sequence of E. coli alkalinephosphatase; SS-b5-T, signal sequence-appended full-length cyto-1 To whom correspondence should be addressed.

2371046-5928/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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KADERBHAI AND KADERBHAI238

esis in E. coli. The b5-T of liver endoplasmic reticulum as recommended by the manufacturer. The standardoperations involving DNA manipulations, i.e., purifi-is a well-characterised, small hemoprotein of 15.5 kDa

which plays a central role in a variety of electron trans- cation, digestion, agarose electrophoresis, and bacterialtransformation, were performed as described pre-fer reactions related to fatty acid desaturation, redox

cycling of estrogen, cholesterol synthesis, and detoxifi- viously (26).The plasmid pA-sct harboring the precursor SS-b5-Tcation of xenobiotics (18,19). The hepatic b5-T is an

amphipathic protein made up of two domains: a solu- gene and bearing the ampicillin resistance gene wasconstructed as follows: plasmid pAA-cyt (24) which ex-ble, enzymically active, heme-containing globular core

(b5) domain comprosed of the first 99 amino acid resi- presses the secretory form of SS-b5 under the controlof phoA promoter was doubly cleaved with XhoI anddues and a smaller membrane-anchoring domain at the

carboxy terminus composed of 35 amino acid residues HindIII. Four staggered segments of double-strandedoligonucleotides coding for the extended portion of b5-(T). B5-T belongs to a class of wide-spread, integral

proteins with a monotopic membrane arrangement in T were synthesised on glass beads using diisopropylphosphoramidite intermediates on an automated Phar-which the globular b5 domain is laterally disposed fac-

ing the cytoplasm (20). Anchorage of b5-T in the endo- macia-LKB Gene Assembler. The deprotected oligonu-cleotides were purified by filtration through NAP-10plasmic reticular membrane is provided by a hydropho-

bic stretch of 22 uncharged amino acid residues in the columns, phosphorylated by the action of polynucleo-tide kinase and repurified using the Wizard DNA puri-T domain (21). The mechanism by which the T domain

targets b5-T specifically to the endoplasmic is presently fication system (Promega, U.K.). Equimolar portions ofthe synthetic oligonucleotides were annealed with theunknown. Our analyses of the membrane insertion se-

quence of b5-T suggested that the T portion contains a larger fragment of XhoI–HindIII-cleaved pAA-cyt andthe duplexes ligated by the action of T4 DNA ligase.25 amino acid residue helical motif which has compara-

ble characteristics to stop-transfer sequences found in Following their transformation and amplification in E.coli TB1, the recombinant plasmids were mapped withthe transmembrane segments of biotopic proteins (22).

Many proteins utilize an N-terminal, cleavable signal XhoI and HindIII restriction endonucleases. The se-quence of SS-b5-T in the plasmid DNA isolated fromsequence in combination with a stop-transfer sequence

to gain their characteristic bitopic membrane disposi- one cell line was confirmed by dideoxy DNA sequencing(27). This E. coli TB1 strain/pA-sct was used in thetion (23). Therefore, to test the functionality of the po-

tential stop-transfer sequence in b5-T, we have con- present study.A 1200-bp DNA fragment coding the full-length ofstructed a gene coding for a fusion form of b5-T carrying

a secretory signal at its N terminus. Previously, we E. coli signal peptidase I gene (including the nativeribosomal binding site) (8) was amplified by PCR. E.have reported that the soluble b5 core, although lacking

a secretable homologue in nature, can be efficiently coli chromosomal DNA and the following pair of PstI-adapted oligonucleotides served as the template andexported from E. coli by appending a signal sequence

(SS) at its N terminus (24,25). In the present study we the primers, respectively.reinstated the T to the C terminus of SS-b5 to investi-gate the outcome of targeting of SS-b5-T in E. coli. Sur- PstIprisingly, the expressed SS-b5-T was overproduced in

primer 1: 5*-TTA CTGCAG AAATAACCCT TAGGAGTTGG-3 *an unprocessed form and selectively targeted to theinner membrane of E. coli. Here we describe the isola- PstItion and characterization of the unprocessed SS-b5-T

primer 2: 5*-GTA CTGCAG A TCTCATAAAT AATTCACGTT-3*precursor. Substrate-level quantities of the SS-b5-Tprecursor protein can be readily purified and it hasproved to be an effective macromolecular substrate for The PCR reaction was performed in a final 50 ml

volume containing 10 mM Tris–HCl (pH 8.8 @ 257C),E. coli signal peptidase.1.5 mM MgCl2, 50 mM KCl, 0.1% (v/v) Triton X-100, 0.2mM each dNTP, 10 mg E. coli chromosomal DNA, 25MATERIALS AND METHODSpmol of each primer, and 2 units Thermus brockianus

DNA Manipulations DNA polymerase. The mixture, overlaid with 50 ml par-affin oil, was subjected to 10 rounds of thermocycling

New England Biolabs (U.K.) and NBL Gene Sciences involving denaturation at 947C for 1 min, annealing at(U.K.) supplied restriction endonucleases and DNA 557C for 2 min, and extension at 727C using a pro-modifying enzymes; the reaction conditions used were grammed Hybaid thermocycler. DNA amplification

was continued for 20 additional cyclings with the an-nealing temperature raised to 627C. The PstI-digested,chrome b5; T, C-terminal tail or membrane anchoring portion of cyto-

chrome b5. agarose-purified, 1200-bp amplified DNA was ligated

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PRECURSOR SUBSTRATE FOR SIGNAL PEPTIDASE 239

into PstI-cleaved plasmid pMCY-ntp (University of AssaysWales Plasmid Bank, Aberystwyth) in an orientation The amounts of SS-b5-T protein present in the biolog-placing the signal peptidase gene downstream of the ical samples were estimated spectrally from the heightspho promoter. The resultant plasmid pMC-spase bear- of the Soret absorption peak at 413 nm for the oxidizeding the ampicillin resistance gene was contained in a form and/or 423 nm for the reduced form using theE. coli TB1 host. millimolar absorption coefficients of 115 mM01

rcm01

and 185 mM01rcm01, respectively (28).

Protein content was estimated by the BradfordCultures method (29) using bovine serum albumin as the stan-dard.The E. coli TB1 strains were maintained and propa-

Precursor processing assays were conducted in a fi-gated in LB liquid or solid media without or with ampi-nal reaction volume of 35 ml (or multiple volumescillin (75 mg/ml). For production of the recombinantthereof) containing 0.4% (w/v) Triton X-100, 50 mMproteins, E. coli strains carrying the relevant plasmidTris–HCl (pH 7.5), 7.15 mM purified SS-b5-T proteinwere batch cultivated for 6 h in a phosphate-limitedand E. coli signal peptidase (amounts specified in the(0.1 mM) MOPS medium (24) containing ampicillin (75text) with incubation at 377C for varying durations.mg/ml); the incubation was at 357C with shaking at 125Reactions were terminated by mixing with 0.25 vol ofrev/min.SDS sample buffer (20% SDS, 75% (w/v) glycerol, 0.05%bromophenol blue) and immediately placing the mix-

Purification of Signal Peptidase tures, contained in tightly capped Eppendorf tubes,over a boiling water bath for 5 min. The proteins pres-We purified the signal peptidase enzyme from theent were separated using a standard 15% polyacryl-membranes derived from 1 liter of the low phosphate-amide gel containing SDS (30). The Coomassie blue-induced E. coli/pMC-spase cells employing the proce-stained gels were densitometrically scanned using adure described by Wolfe et al. (5).Sharp JX325 scanner and the protein profiles quanti-fied using Phoretix 1D software (Phoretix, U.K.) op-erating under MS Windows 3.1. Since the knownPurification of SS-b5-T Proteinamount of the precursor protein used at the start of

The following procedures were performed at 47C. E. the reaction was known, product formation at a givencoli/pA-sct cells harvested (5000g 1 5 min) from 1 liter time point was determined from comparison of the inte-of culture were resuspended in 10 ml of 10 mM Tris– grated SS-b5 (corrected for the cleaved signal peptide)HCl (pH 8), 1 mM EDTA (TE) containing 10 mM bovine and SS-b5-T band areas.heme. The cells were treated with 0.1 mg lysozyme/ml

Laser Desorption Mass Spectroscopyand left to stand on ice for 30 min. Lysis was promotedby subjecting the suspension to 10 1 15-s sonications The mass spectra of SS-b5-T and b5-T were generated(Soniprep 150, MSE) with a 20-s interval between each using a Perspective Biosystems laser desorption masspulse. The membranes were prepared from a post-cel- spectrometer (40). After extensive dialysis againstlular fraction (1000g for 20 min) by centrifugation at 0.1% Na deoxycholate, the protein samples were mixed105,000g for 1 h, resuspended in 2.5 ml TE buffer, and with an equal volume of a matrix solution composed ofgradually added to 50 ml of 40% (v/v) acetonitrile while sinapinic acid saturated in CH3CN:water (1:3 (v/v)).stirring in a beaker. A low-speed (8000g for 30 min) The dried mixture was irradiated with 337 nm lasersupernatant fraction representing the recombinant light in order to destroy the matrix and the samplehemoprotein extract was diluted with an equal volume accelerated in a voltage field of 28 kV. The mass wasof 25 mM Tris–acetate (pH 8) (TA) and applied onto a evaluated by timing the flight of the released sampleTA-preequilibrated DEAE Sepharose CL-6B column (1 to the detector. The sizes of proteins were derived fromcm i.d. 1 10 cm high). The column was washed with the deconvoluted mass spectra.100 ml TA and the bound hemoprotein was then eluted

Protein Sequencingas a broad peak during the application of a linear NaClThis was carried out according to the manufacturersgradient (0 to 0.5 M) in TA containing 1% (w/v) NP-40.

instructions on an Applied Biosystems Model 470A gasHemoprotein-enriched fractions were pooled, exten-phase sequencer.sively dialyzed against TA, and rechromatographed on

an HR5/5 Mono Q column (Pharmacia-LKB, U.K.) us-RESULTSing an automated Pharmacia-LKB FPLC system, fol-Expression of SS-b5-T in E. colilowing the procedure essentially as described above.

Passage of the final preparation through an Amberlite Figure 1A shows the schematic map of the gene con-struct coding for the tripartite SS-b5-T protein. TheXAD-2 resin column depleted residual NP-40.

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KADERBHAI AND KADERBHAI240

tion of a hemoprotein within the bacterial cells. Thiswas substantiated by SDS–PAGE analyses of total pro-tein fractions from the induced bacteria which showeda time-dependent, massive accumulation of a 21-kDapolypeptide (Fig. 2B) that strongly and specificallycross-reacted with sheep anti-b5 polyclonal antibodies(data not presented). Thus, amounts of the 21-kDapolypeptide throughout bacterial growth correspondedclosely with the changes in the induced hemoproteindetected spectroscopically.

Purification of 21-kDa Hemoprotein

Electrophoretic analysis of the bacterial subcellularfractions showed that the 21-kDa protein was almostentirely present in the (cytosolic) membrane fractionwith insignificant amounts detectable in the remainingsoluble fraction (Fig. 3). The hemoprotein content inthe isolated membranes was enriched threefold overthat estimated in the total cell lysates (Table 1). Intheir physical association with the lipid bilayer, boththe 21 kDa and hemoprotein appeared to be integralcomponents since neither was extractable by treatmentof the isolated membranes with 0.1 to 1 M NaCl or 0.1 M

Na2CO3 (data not shown). Treatment of the membraneswith 1% (w/v) Nonidet P-40 efficiently extracted thehemoprotein but the solubilization initiated a time-de-FIG. 1. (A) Schematic map of the gene coding SS-b5-T. phoA, pro-pendent loss of the 21-kDa protein with concomitantmoter; S/D, Shine–Dalgarno sequence; SS, alkaline phosphate signal

sequence; b5 and T, globular and tail portions of cytochrome b5, re- appearance of an 18-kDa component (data not pre-spectively. (B) Amino acid sequence of SS-b5-T. The italicized and sented) which was later identified as a derivative lack-bold residues denote, respectively, the SS and the T portions of the ing the SS portion. An alternative approach of treat-fusion protein.

ment of membranes with 40% (v/v) acetonitrile resultedin extraction of the 21-kDa protein in an intact formwith solubilization of about 50% of the membrane he-fusion protein (Fig. 1B) sequencially composed of the moprotein (Fig. 3, lane 5). After loading onto the DEAE-21 residues of alkaline phosphatase SS, one remnant Sepharose CL-6B column and development with a saltN-terminal residue of alkaline phosphatase, 99 resi- gradient, the hemoprotein eluted at 300 mM NaCl asdues of b5, and 35 residues of T. The b5-T portion of a well-defined peak, representing a 78% hemoproteinthe chimera represents the full-length endoplasmic re- purity (Table 1). Further purification by Mono Q chro-ticulum cytochrome b5 of rat liver. The fusion was cre- matography on an FPLC system separated a single ma-ated by an in-phase linkage of the synthetic T DNA jor peak representing a hemoprotein at a specific con-with SS-b5 in the plasmid pAA-cyt which was pre- tent of 94% (Fig. 3, lane 7) from some minor impuritiesviously reported (24) to direct high-level secretion of of lower molecular mass. Typical yields of the purifiedthe b5 protein into the periplasm of E. coli. The place- 21-kDa protein were around 5 mg protein/liter of cellment of the SS-b5-T sequence downstream under the culture at a cell density of Ç2 O.D. at 600 nm.control of the E. coli phoA promoter assures the gene

is tightly repressed during growth of the bacteria inCharacterization of the 21-kDa Protein as SS-b5-Ta standard rich medium. However, the gene can be

optimally expressed in E. coli cultured in the Mops The isolated 21-kDa protein was subjected to spectralanalyses in order to investigate its relatedness to na-medium containing low (0.1 mM) phosphate.

When E. coli TB1 cells harboring the plasmid pA-sct tive b5-T (Fig. 4). The 21-kDa protein had Soret bandat 413 nm in the oxidized state, and on reduction, shiftwere cultured in the phosphate-limited Mops medium,

they displayed an absorption spectrum characteristic in absorbance to 423 nm was observed with emergenceof peaks at 555 and 527 nm. The absorption spectrumof a b-type hemoprotein in the reduced state. The obser-

vation that the magnitude of the Soret absorption peak of the oxidized versus reduced forms of the recombinanthemoprotein was identical to the trypsin-digested b5at 423 nm (Fig. 2A) increased concomitantly with cul-

ture growth indicated de novo synthesis and accumula- purified from mammalian tissue, in having a character-

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PRECURSOR SUBSTRATE FOR SIGNAL PEPTIDASE 241

FIG. 2. Production of the 21-kDa protein in E. coli. (A) Spectral detection of the hemoprotein in intact bacteria. (B) Electrophoreticdetection of the 21-kDa fusion protein in E. coli lysates by staining of the gel with Coomassie blue; the arrow marks the position of theinduced band.

istic maximum at 423 nm and a minimum at 409 nm. nine was found to be unprocessed in the synthesisedproduct.The absorption coefficients determined for the oxidized

and reduced forms of the 21-kDa hemoprotein at the The presence of T sequence in the 21-kDa proteinvarious absorption peaks were close to the values pre- was indicated by comparison of its electrophoretic mo-viously reported for the mammalian liver and erythro- bility with the related derivatives, i.e., SS-b5, b5-T, andcyte b5 (28). b5. In accord with their relative sizes the mobilities

were SS-b5-Tú b5-Tú SS-b5ú b5. Processing of the 21-A preparation of the purified 21-kDa protein wassubjected to 10 rounds of automated peptide degrada- kDa protein by signal peptidase (see below) converted it

to a form which electrophoretically migrated with ation. This yielded an N-terminal amino acid sequenceidentical to that deduced from the nucleotide sequence mobility identical to that of b5-T. The mass of SS-b5-T

determined by laser desorption spectroscopy yielded aof SS except that the GTG-encoded initiator methio-

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KADERBHAI AND KADERBHAI242

FIG. 4. Spectral characteristics of SS-b5-T. Oxidised (O), dithionite-FIG. 3. Purification of the 21-kDa protein. Protein loadings:reduced (R) and reduced versus oxidised (R-O) spectra of a 20 mgmarker proteins, 25 mg; total cellular fraction, 75 mg; cytosolic frac-purified preparation of SS-b5-T.tion, 75 mg; membranes, 60 mg; acetonitrile extract, 60 mg; Sepharose

CL-6B eluate, 12 mg; Mono Q chromatographic peak, 4 mg.

precursor processing complied with the reaction speci-ficity of signal peptidase (6,9). Processing was virtuallymolecular weight of 17,665 that agreed well with theunaffected by any of the following commercially avail-calculated molecular mass 17,669. These findings es-able protease inhibitors of serine-, metallo-, thiol-, andtablished the identity of the 21-kDa protein as beingaspartyl-proteases: 2.5 mM 4-(aminophenyl)-methane-the SS-b5-T protein.sulphonyl fluoride, 0.15 mM pyridyldisulfide, 1 mM

benzamidine, 50 mM aprotinin, 0.5 mM phenylmeth-Processing of SS-b5-T by Signal Peptidase anesulfonyl fluoride, 1 mM 1,10 o-phenanthroline, 1 mM

e-amino-n-caproic acid, 10 mM dithiothreitol, 2 mM N-Incubation of a 50-fold molar excess SS-b5-T with thepurified signal peptidase resulted in a time-dependent a-tosyl-L-lysine-chloromethylketone, 2.5 mM leupeptin,

2.5 mM iodoacetamide, 10 mM b-mercaptoethanol, 2.5loss of the 21-kDa band with concomitant formation ofan 18-kDa band that was almost quantitatively recov- mM Na2 EDTA, 100 mM antipain dihydrochloride, 150

mM chymostatin, 250 mM bestatin, 5 mM pepstatin, 2.5ered at the end of the reaction (Fig. 5). The processedprotein was immunoelectrophoretically identical with mM n-carboxybenzoxy-L-phenylalanine chloromethyl

ketone. The catalysis was unaffected when the enzymenative b5-T from rat liver (data not shown). The massspectroscopically determined molecular mass of 15,516 was chemically modified or cross-linked by ethyl-3-(3-

dimethyl-) aminopropyl-carbodiimide prior to assay-of the signal peptidase-processed product matched wellwith the calculated size (15,514) of (arg)-b5-T. More- ing. However, the Group II heavy metal ions Pb2/,

Cu2/, Zn2/, Mg2/, Fe2/, Mn2/, and Co2/ proved particu-over, the N-terminal sequence of the signal peptidase-processed product of SS-b5-T yielded the predominant larly inhibitory at concentrations ranging from 5 to 10

mM. HgCl2 was particularly potent inhibitor with 50%sequence as (Arg)–Met–Ala–Glu–Gln, indicating that

TABLE 1

Purification of SS-b5-T

Volume Protein Hemoproteina Hemoprotein yield Hemoprotein specific PurificationFraction/step (ml) (mg) (mg) (%) content (%) (fold)

Homogenateb 10 935 49 100 5 1Membranes 3 148 26 52 17 3Acetonitrile extract 52 54 13 26 24 4.5DEAE-Sepharose 20 13 10 20 78 15FPLC Mono-Q 3 6.4 6 12 94 18

a Spectrally estimated.b Cell lysate from 1 liter culture.

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PRECURSOR SUBSTRATE FOR SIGNAL PEPTIDASE 243

ature-induced phase separation (32) of the membranesor the isolated SS-b5-T protein (data not presented).

The crystal structure of the 86 residue globular bo-vine b5 has been reported at a 2.8A resolution (33).In common with all of the sequenced mammalian b5

homologues, the bovine b5 is highly conserved in itsprimary structure (21). A large proportion of the b5

molecule (residues 26 to 83) is devoted to the formationof a crevice to accommodate the heme which fits tightlyinto the hydrophobic lining of the pocket. The two histi-dines at positions 45 and 68, located in the nonhelicalloops at the top of the crevice, extend into the centerof the pit to bind the heme iron. The heme-linked histi-dine residues are held rigidly by a variety of intermolec-ular forces and cannot be displaced from the iron atomby other ligands without disrupting the protein struc-ture. Since the spectral properties of SS-b5-T proteinare indistinguishable from either b5 or b5-T, it is rea-sonable to deduce that the b5 domain must be fullypreserved in the fusion protein. In this respect it ispossible to gain an insight into the likely, higher or-dered structure of the SS-b5-T chimera by modeling onthe known crystal structure backbone of b5. The b5 isa compacted domain the interior of which is distinctlyapolar and the exterior is extensively combed with sidechains of charged and hydrophilic amino acids. Thesurface-exposed residues near the amino and carboxyends are thought to have no apparent structural roleand form flexible linkages. With these insights, we ex-FIG. 5. Processing of SS-b5-T by signal peptidase. The assays were

performed for varying periods as described under Materials and tended the surface-exposed termini of b5 with the pre-Methods. The concentrations of the substrate and enzyme were 7.15 dicted structures (34) of the (i) amino-terminal SS andand 0.14 mM, respectively. Extent of cleavage (top) monitored electro- (ii) carboxy-terminal T portions (35). In this structurephoretically and (bottom) quantitated densitometrically from (top).

(Fig. 7) the b5-T linkage has a flexible, randomly coiledsegment which demarks the zone between the largercompact b5 domain and the lipophilic tail; this is the

inhibition at 0.5 mM. The effect of varying the amounts trypsin-susceptible region of the membrane-bound b5-of signal peptidase on the rate of SS-b5-T processing at T that enables proteolytic generation of a soluble b5the fixed concentration of 7.2 mM is shown in Fig. 6. form (36). Likewise, the protrusion of the amino-termi-Cleavage was sufficiently slowed down at the substrate nal SS linkage in the model is based on the premiseto enzyme ratio of 800 to yield about 50% cleavage after that the SS is clearly accessible for both the recognition1 h. At substrate:enzyme ratios of 1800 or above the and the precise cleavage by the bulkier signal peptidaserates of SS-b5-T cleavage were linear within the first molecule. Both the lipophilic termini extensions have30 min. a high propensity to form a-helices which would ther-

modynamically favor their positioning away from thehydrophilic b5 core. Three-dimensional modeling of the

Structure of SS-b5-T SS-b5-T protein on Hyperchem Molecular Modeller V3indicated that the terminally exposed hydrophobic seg-A hydropathy plot of the SS-b5-T primary structurements in the tripartite protein possess sufficient flexi-based on the algorithms described by Kyte and Doolit-bility and dimensions to establish interactions via theirtle (31), with a moving average window size of five central hydrophobic cores and the terminal ionicresidues, displayed the distinctive tripartite amphi- groups. Moreover, the adjoining hydrophobic a–a heli-pathic structure. The plot showed that the soluble hy- cal cylinders are of a length sufficient to traverse the

drophilic b5 core was flanked on both sides by the mem- lipid bilayer.brane-seeking, hydrophobic amino- and carboxy- termi-

DISCUSSIONnal-linked SS and T segments. This amphipathiccharacter of the SS-b5-T protein was evidenced by its In this study we have shown that SS-b5-T expression

in E. coli resulted in substrate level production of thecomplete recovery with Triton X-114 following temper-

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KADERBHAI AND KADERBHAI244

FIG. 6. The effect of varying signal peptidase concentration on the cleavage of SS-b5-T. The reactions were performed for 60 min at theenzyme concentrations ranging from 2 to 20 nM with the concentration of substrate fixed at 7.15 mM. Extents of processing (top) monitoredelectrophoretically and (bottom) quantitated from densitometric traces.

unprocessed SS-b5-T protein. Following 6 h of growth by the conventional anion-exchange chromatography.The authenticity of the isolated SS-b5-T has been veri-as much as 50 mg of SS-b5-T protein was produced per

liter of batch-cultivated E. coli, without the formation fied by a variety of criteria including N-terminal se-quencing, laser desorption mass spectroscopy, immu-of inclusion bodies. The SS-b5-T protein was specifically

integrated with the cytoplasmic membranes where it noelectrophoretic analyses, phase separations, spectro-photometric analyses, and its processing by purifiedrepresented about 5% of the total bacterial protein.

Only a proportion (35%) of the bacterially produced SS- signal peptidase.The fact that SS-b5-T was spectrally indistinguish-b5-T existed in its holoform. However, supplementation

of the lysed cells with exogenous bovine heme con- able from the native hemoprotein is indicative of thestructural retention of the tightly folded heme-bindingverted the apo-protein pool to holocytochrome. The

presence of a significantly large pool of the apo-SS-b5- globular domain which must, therefore, have been cor-rectly folded and preserved against likely perturba-T within the cells could be attributed to the limited

capacity of the E. coli host to synthesize the prosthetic tions by the neighboring SS and the native T peptideextensions. Molecular modeling of SS-b5-T on Hyper-heme group (37) during overproduction of the recombi-

nant protein. chem 3, based on combined data derived from predic-tion methods and the crystal structure of b5, supportsA procedure has been developed for the isolation of

the intact SS-b5-T protein. This procedure involves ex- this postulated amphipathic structure of the chimerain which a spatially distinct hydrophobic core is formedtraction of the membranes with acetonitrile which solu-

bilises SS-b5-T in an undegraded form. In turn the ex- by apolar interactions between the T and the SS seg-ments. Based on thermodynamic considerations, thetracted SS-b5-T can be isolated in a homogeneous state

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PRECURSOR SUBSTRATE FOR SIGNAL PEPTIDASE 245

many transmembrane proteins (23). It is plausible thatthe hydrophobic interactions between the helical por-tions of the T and SS within the lipid phase, and possi-bly between the charged residues adjacent to the mem-brane phase (Fig. 7), may have displaced the processingsite, rendering it to be inaccessible to signal peptidase.However, this apparent inhibition was overcome by de-tergent treatment of the membranes. This model raisesa strong possibility that SS cleavage of precursors bysignal peptidase is an event that could occur after thepolypeptide is almost fully translocated. Our postu-lated topography of SS-b5-T in the inner membraneremains to be investigated, employing selective chemi-cal modification and progressive proteolysis ap-proaches.

The isolated SS-b5-T is a macromolecular substratethat is faithfully and efficiently processed by the signalpeptidase. The specificity of the cleavage reaction isFIG. 7. Potential structure of SS-b5-T. The schematic backboneverified by N-terminal sequence of the processed pro-structure of the three domains is shown; charged residues are shown

in the SS and T segments. The crystal structure-derived backbone tein band which matches the expected maturation siteof the globular b5 domain extends from the amino acid residues 8 to of the precursor in vivo. The cleavage of SS-b5-T by86 (34). The rectangle encompasses the probable regions of the SS signal peptidase being insensitive to all the classic pro-and T helices that traverse the lipid bilayer. Signal peptidase cleav-

tease inhibitors is yet another indicator of the unusualage site is denoted by the arrow.reaction catalyzed by this endopeptidase (10–12). As aprecursor protein that is expressible and isolatable inabundant quantities in a unique ‘‘soluble’’ form, SS-b5-hydrophobic segments must protrude away from the

charged surface of the globular b5 and function to dou- T can be exploited as a nonlabeled reagent for deci-phering the reaction mechanism of the signal peptidasebly anchor the protein in the membrane. Like many

rod-shaped proteins (26), the anomalous, slower elec- and as a probe for exploring events in protein transloca-tion.trophoretic mobility of the recombinant SS-b5-T re-

solved under the denaturing effect of SDS may arisefrom preservation of the head-tail interactions im- ACKNOWLEDGMENTparting a decreased level of detergent binding (38).

This work was supported by a grant from the University of WalesThe mode of SS-b5-T targeting to integration andResearch Funds. We are most grateful to Prof. M. Akhtar and Petereventual organization in the membrane remains in- Robichaud for the mass spectroscopic measurements.

triguing as the SS cleavage site appears to be inaccessi-ble to the signal peptidase unless the membrane integ-

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