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Toxicon 58 (2011) 644–663

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Toxicon

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Review

Scorpion and spider venom peptides: Gene cloning and peptideexpression

V. Quintero-Hernández a, E. Ortiz a, M. Rendón-Anaya a, E.F. Schwartz b, B. Becerril a,G. Corzo a,*, L.D. Possani a,*a Instituto de Biotecnología - UNAM, Avenida Universidad, 2001, Colonia Chamilpa, Apartado Postal 510-3, Cuernavaca, Morelos 62210, Mexicob Laboratório de Toxinologia, Departamento de Ciências Fisiológicas, Universidade de Brasília, Brasília, DF, Brazil

a r t i c l e i n f o

Article history:Received 8 July 2011Received in revised form 8 September 2011Accepted 22 September 2011Available online 28 September 2011

Keywords:ESTExpressionGene cloningScorpionSpiderToxin

* Corresponding authors. Tel.: þ52 77 73171209; fE-mail addresses: corzo@ibt.unam.mx (G. Corzo

mx (L.D. Possani).

0041-0101/$ – see front matter � 2011 Elsevier Ltddoi:10.1016/j.toxicon.2011.09.015

a b s t r a c t

This communication reviews most of the important findings related to venom componentsisolated from scorpions and spiders, mainly by means of gene cloning and expression.Rather than revising results obtained by classical biochemical studies that report structureand function of venom components, here the emphasis is placed on cloning and identi-fication of genes present in the venomous glands of these arachnids. Aspects related tocDNA library construction, specific or random ESTs cloning, transcriptome analysis, high-throughput screening, heterologous expression and folding are briefly discussed,showing some numbers of species and components already identified, but also shortlymentioning limitations and perspectives of research for the future in this field.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

The arthropods constitute one of the most abundantand widely distributed groups of animals on earth. Scor-pions and spiders, generally called arachnids, are importantrepresentatives of this group of living organisms because oftheir large number of species, estimated to be in the orderof 1600 for scorpions andmore than 39,000 for spiders, butalso because they contain potent venoms used for feedingand defense. The estimated number of possible compo-nents present in their venoms exceeds by far the value ofseveral millions (Escoubas and Rash, 2004). The venomsfrom those that are dangerous to humans have beenintensively studied, showing that they have a great varietyof peptides and enzymes with interesting properties. Theinitial work was focused at species dangerous to humans,

ax: þ52 77 73172388.), possani@ibt.unam.

. All rights reserved.

but more than 98% of the species are still completelyunknown concerning the types of components present intheir venoms. For scorpions and spiders, there are severaltypes of toxic peptides described that affect mainly cellularcommunication. They recognize ion channels, causingblockage or modification of their open and closing mech-anisms, provoking an anomalous depolarization of cells(reviewed in Catterall et al., 2007). Additionally, enzymessuch as hyaluronidase, phospholipase and proteases havebeen reported (reviewed in Possani et al., 1999; Escoubasand Rash, 2004). Small peptides with antimicrobial andanti-parasitic activities or other pharmacological actionshave also been reported (Conde et al., 2000; Torres-Larioset al., 2000; Corzo et al., 2002; Verano-Braga et al., 2008).Scorpions also have lipolytic enzymes, metalloproteinasesand lysozime activities (Soudani et al., 2005; Fletcher et al.,2010). The chemical composition of spider venoms isdiverse ranging from low molecular weight organiccompounds such as acylpolyamines to complex peptidesand proteins. Although the acylpolyamines represent the

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vast majority of organic components from the spidervenom, spider peptides act specifically; they can discrimi-nate Ca2þ, Naþ and Kþ ion channel subtypes and are able tomodulate ionic currents in ligand- and voltage-gated ionchannels. The peptidic fractions containing amino acidsrepresent approximately two thirds of the dryweight of thespider venom (Corzo et al., 2002).

Most scorpion and spider venom components havebeen identified and characterized using milked venomfrom living specimens. Individual components were iso-lated and separately characterized concerning their struc-ture and function (reviewed in Grishin, 1999; Goudet et al.,2002; Rodríguez de la Vega and Possani, 2004, 2005; Sollodet al., 2005; Rash and Hodgson, 2002; De Lima et al., 2007;Gordon et al., 2007). However, nowadays a more modernstrategy is been followed by the use of mass spectrometryanalysis and mainly molecular biology techniquesinvolving gene cloning and sequencing.

Starting from 1989, when the first report of a scorpiontoxin cDNA was published (Bougis et al., 1989), the use ofmolecular biology tools in the indirect characterization ofthe different peptides and proteins expressed in scorpionsand spiders has gained momentum. Hundreds of precursorsequences have been reported to date. The GeneBank nowcontains more than 4300 scorpion nucleotide sequences; ofthem some 1900 being expressed sequence tags (ESTs) andmore than 38,000 spider nucleotide sequences (with morethan 32,000 ESTs). These data easily outnumber those ob-tained from standard biochemical procedures involvingpurification, mass spectrometry (MS) analysis andsequencing of individual peptides and proteins (Pimentaet al., 2003). Apart from the universality and plasticity ofthe cloning methods, several other advantages stand out.Genome librariesordirect cloning fromgenomicDNAallowsassessing the gene structure, the intron/exon compositionand the regulatory sequences functioning at the transcrip-tion level. On the other hand, cDNA libraries, give valuableinformation regarding the organization of the non-matureproteins, including the presence of a signal peptide, a pre-and/or a pro-peptide and sometimes even about C-terminalamidation, post-translational modifications that arefrequently impossible to determine from the profile ofmature proteins. cDNA can be fractioned by size prior tolibrary construction, allowing the researcher to enrich thelibraries either in small peptide or in large protein-encodinggenes. This samestrategycanbeused to increase thechancesof cloning sequences from less abundant mRNAs when theinvolved tissue has several major overexpressed compo-nents, as is frequently the casewith the venomglands. Thereis another very significant advantage of the cDNA cloning:since mature mRNAs fromwhich cDNA is copied contain nointrons, the cDNA sequences can be used for expression ofthe desired peptides or proteins in heterologous systems.Thus, an indirect benefit of cDNA sequence explorationsresides in the possibility of having a permanent and reliablesource of the product of interest, once the protocol ofexpression and folding is correctly established, obviating theneed of organism removal from the ecosystems.

In this review we will focus on the evolution of thecloning methods used to generate sequence informationfrom scorpions and spiders, and the results obtained. We

will move from direct cloning of specific sequences to cDNAlibraries amplified fromtissues containing thevenomglandsand then, to massive sequencing of cDNA libraries thatconstitute a new level of information: the transcriptome.Wewill also explore the different expression/folding systemsused for the generation of functional peptides.

2. Cloning of cDNAs encoding scorpion and spidervenom gland peptides

For decades, the study of venomous animals has focusedon the identification and biochemical characterization ofspecific toxins that have medical or pharmacologicalimportance; however, little is known regarding the cellularprocesses that take place within the venom glands duringthe assembly of the toxic arsenal. In recent years, different“omic” approaches have become a very powerful tool forunderstanding the complexity of venomous animals. Tran-scriptomics in particular, has been widely used to explorethe transcriptional diversity of venom glands of severalAraneomorphae and Mygalomorphae spiders as well as ofscorpion species belonging to the Buthidae, Scorpionidae,Euscorpiidae, Liochelidae and Iuridae families.

2.1. cDNAs from scorpion venom glands

Two venom glands are located in a very well delimitedcompartment in the scorpion’s body: the last post-abdominal segment called telson. It is in these glands thatthemRNAs encoding for all the peptide venom componentsare transcribed. It is therefore relatively simple to use thislast metasoma segment as a biological sample to gather thegenetic information regarding the venom components.Using molecular biology techniques, such as RT-PCR, cDNAhas been amplified from total mRNA purified from telsonmacerates. From this cDNA several peptide precursorsequences have been identified, with the use of oligonu-cleotides which sequences were derived from the knownpeptide sequences, obtained by direct Edman degradation.

Thefirst report of a scorpionpeptide precursor-encodingsequence successfully cloned was that of AaHII, the mostpotent neurotoxin from the venom of the North Africanscorpion Androctonus australis Hector (Bougis et al., 1989).This, together with the precursors for several other toxinsfrom the same scorpion that affect mammals and insects,were isolated from a cDNA library constructed from themRNA obtained from a macerate of 30 A. australis telsons.Oligonucleotides designed from the AaHII peptide sequencewere used as hybridization probes to identify the completeprecursor from 400,000 colonies of the cDNA library.

The molecular characterization of the first venomcomponent-encoding ESTs showed that the precursorswere larger than the mature toxins purified from thevenom, and that larger amino acid sequences are encodedin the mRNAs, both at the 50 and 30 corresponding regions.The sequence analysis revealed that the precursors hadabout 20 amino acid-long signal peptides and some extraresidues at the carboxyl terminus, whichwere absent in themature toxins. Amidation of the C-terminal amino acidsmight occur postranslattionaly (Bougis et al., 1989; Becerrilet al., 1993, 1996).

V. Quintero-Hernández et al. / Toxicon 58 (2011) 644–663646

In order to determine the sequence encoding theprecursor of the insect-specific toxin BjIT2 from the venomof the scorpion Buthotus judaicus, a cDNA library wasprepared from the venom gland mRNA and used for PCRamplification with specific oligonucleotides, designedbased on the amino acid sequence of the BjIT2 toxin, asprimers (Gurevitz et al., 1990). This strategy has been andstill is the most widely employed for the isolation ofprecursor-encoding sequences (Table 1). This PCR techniquecan also be implemented with cDNA libraries as template,instead of just cDNA. Several precursor sequences have beendescribed with this last method for the Centruroides noxiusHoffmann toxins (Becerril et al., 1993; Vazquez et al., 1995).

Genomic DNA has also been used as template togetherwith specific oligonucleotides, which has resulted in thecloning of the DNA sequences coding for several Naþ

channel toxins from the species Tityus (T.) serrulatus, Tityusbahiensis and Tityus stigmurus (Becerril et al., 1996).

Nowadays, cDNA and genomic DNA are still used forprecursor search but the complete sequences of theprecursors are determinedwith the help of the RACE (RapidAmplification of cDNA Ends) technique, which allows toclone full-length cDNA sequences when only a partialsequence is known. Typically, first strand cDNA is synthe-sized from total RNA or mRNA and then an adaptamer isligated to the 30 end of the cDNA. An oligonucleotide specificfor the adaptamer is then used in conjunctionwith a primerfor the known sequence (or derived from the sequence ofthe mature peptide of interest) to amplify the completeregionbetween the adaptamerand thegene-specific primer(50-RACE) or a gene-specific primer is used together with anoligo(dT) to amplify in the other direction (30-RACE). Theprecursors for neurotoxins BmK AS and BmK AS-1, BmP01,BmP03, BmP05 and the insect-specific BmK IT-AP, all fromscorpion Buthus martensi Karsch, were the first to be clonedusing the 50-RACE and 30-RACE techniques (Lan et al., 1999;Wu et al., 1999; Xiong et al., 1999). A different strategy wasemployed to isolate the coding sequence for the anatoxinTsNTxP from the scorpion Tityus serrulatus. A clonewith thecomplete cDNAwas identified from an expression library ofcDNA using anti-TsNTxP antibodies (Guatimosim et al.,1999). To date, more than a hundred ESTs and genesequences have been cloned using the described method-ologies, including phospholipases, chlorotoxin, thebradykinin-potentiating peptide (Bpp), the anti-epilepsypeptide BmK AEP, neurotoxins, insect toxins, Naþ channel-specific toxins, Kþ channel-specific toxins, Ca2þ channel-acting peptides, antimicrobial peptides, cytochromeoxidases and analgesic/antitumor peptides (Table 1).

The genomic transcriptional units of different scorpiontoxins have also been reported. AaHI, from A. australis,which recognize sodium channels; kaliotoxin 2 fromA. australis, BmKTX, BmTX1 and BmTX2 from B. martensiKarsch, active on Kþ channels; BmP01, BmP03 and BmP05from B. martensi Karsch, active on small conductanse Ca2þ-activated potassium channels, and Bm-12, a chlorotoxin-like peptide from B. martensi Karsch, all share the samestructural components: two exons and a single intron(Delabre et al., 1995; Legros et al., 1997; Dai et al., 2000;Wuet al., 1999, 2000) and have identical genomic organization:the transcriptional unit starts with the first exon which

encodes for the amino terminus of the signal peptide, thencomes the intron, followed by the second exon, whichencodes the carboxyl terminal region of the signal peptideand the complete mature peptide. Three genes, BmKa1,BmKa2 and BmKb1, that code for non disulfide-bridgedpeptides from the Chinese scorpion Mesobuthus martensi,show distinct genomic structural patterns (Luo et al., 2005).The BmKa1 gene does not show any introns in its sequence,whereas the BmKa2 gene is composed of two exons,interrupted by a 67 bp intron that is located in the maturepeptide region. Two genomic homologs of the BmKb1cDNA sequence, named BmKb1’ and BmKb2, were shownto have an intron at the signal peptide. The genes encodingfor two potassium channel toxins, BmKalphaTx11 andBmKalphaTx15, have also been analyzed (Xu et al., 2005).Their sequence showed that a 500 bp long intron interruptsthe signal peptide region of both toxins. Finally, thegenomic characterization of the BmCa1 toxin (Zhijian et al.,2006b) revealed that it consists of three exons separated bytwo introns, one at the end of the signal peptide region(72 bp long) and the other in the sequence correspondingto the mature peptide (1076 bp long).

2.2. cDNAs from spider venom glands

The first molecular characterization of a spiderprecursor sequence was that of the Ca2þ channel-blockingheterodimeric u-agatoxin IA from a cDNA library from thevenom gland of Agelena aperta. The transcript for u-aga-toxin IA was detected by colony hybridization andsequenced using the Maxam-Gilbert method (Santos et al.,1992). Presently the reverse transcription polymerase chainreaction (RT-PCR) technique has been commonly used toproduce easy, fast and reliable DNA copies of an RNAtemplate. RT-PCR has been applied for cloning spider toxincDNAs, allowing to conserve spider venom resources and toaccess the complete set of mRNAs coding for venom spiderpeptides. Krapcho and collaborators used for the first timeRT-PCR to sequence the insecticidal toxin DTX9.2, from thevenom of the spider Diguetia canities (Krapcho et al., 1995).The cDNA encoded a 94 amino acid precursor, which wasprocessed to the active mature peptide by removal of thesignal and pro-peptide sequences. Moreover, a genomiclibrary was constructed and the gene for DTX9.2 was iso-lated showing that the transcriptional unit spans 5.5 kilo-bases and is composed of five exons. DNA sequencesupstream from the first exon contain a TATA box and twopalindromic sequences (one with homology to a CAATconsensus), which together may constitute a functionalpromoter. The highly segmented gene structure observedin the insecticidal toxin DTX9.2 suggested that a mecha-nism, such as exon shuffling, might play a role in theevolution of spider venom peptides (Krapcho et al., 1995).

3. ESTs from cDNA libraries

3.1. Specific screening of ESTs from scorpion venom glands

Venom gland transcriptomes have been extensivelyused for direct search of toxin sequence precursors usingspecific oligonucleotides. This was the strategy adopted for

Table 1cDNAs from scorpion venom glands.

Peptide Funtion Scorpion species Template or/and method Reference

Toxin II Neurotoxin Androctonus australis cDNA library, usingoligonucleotide probes

Bougis et al., 1989

BjIT2 Depressant insect selectiveneurotoxin

Buthotus judaicus cDNA and oligonucleotideprimers prepared on basisof the known amino acidsequence of the depressantinsect toxin II

Gurevitz et al., 1990

LqhIT2 Depressant insect selectiveneurotoxin

Leiurus quinquestriatushebraeus

cDNA and specific primers Zlotkin et al., 1993

STox II-10 CnH toxins (STox) Centruroides noxiusHoffmann

cDNA library and specificprimers

Becerril et al., 1993

Cn1 and Cn2 Naþ channel-specific toxins Centruroides noxiusHoffmann

cDNA library and specificprimers

Vazquez et al., 1995

Toxins g-b, III-8b, IV-5b,g-st and III-8st

Naþ toxins Tityus bahiensis andTityus stigmurus

Genomic DNA andoligonucleotidessynthesized accordingto known cDNA sequencesof toxin gamma

Becerril et al., 1996

Toxin IV-5 Toxin Tityus serrulatus cDNA and degeneratedprimers

Corona et al., 1996

IpTxi Imperatoxin I, Ca2þ toxin Pandinus imperator cDNA library anddegenerated primers

Zamudio et al., 1997

BmK M1 Neurotoxins Buthus martensii Karsch cDNA library anddegenerated primers

Xiong et al., 1997

TsNTxP Non-toxic protein,immunogen

Tityus serrulatus cDNA expression libraryusing anti-TsNTxP antibodies.

Guatimosim et al., 1999

BmK AS and BmK AS-1 Neurotoxins Buthus martensi Karsch 30 and 50 RACE (RapidAmplification of cDNA Ends)

Lan et al., 1999

BmP01, BmP03, BmP05 Neurotoxins Buthus martensi Karsch cDNAs, 30- and 5’-RACE Wu et al., 1999BmK IT-AP Excitatory insect

selective toxinButhus martensii Karsch 30 and 50 RACE (rapid

amplification of cDNA ends)Xiong et al., 1999

BmTXKbeta andBmTXKbeta2

TXKbeta toxins Buthus martensii Karsch cDNA library anddegenerated primers

Zhu et al., 1999

Phospholipase A2 Phospholipase Pandinus imperator cDNA library anddegenerated primers

Conde et al., 1999

Bm-12 Insect neurotoxin Buthus martensi Karsch 30 and 5’RACE (RapidAmplification of cDNA Ends)

Wu et al., 2000

Scorpine Anti-malaria andanti-bacterial agent

Pandinus imperator cDNA library anddegenerated primers

Conde et al., 2000

BmKbpp Bradykinin-potentiatingpeptide (Bpp)

Buthus occitanus cDNA Zeng et al., 2000

BeKm-1 Specific blocker of hERG1potassium channels.

Buthus eupeus Rapid amplification ofcDNA ends polymerasechain reaction techniquefrom mRNA (RACE)

Korolkova et al., 2001

BmK AEP BmK anti-epilepsy peptide Buthus martensi Karsch cDNA, 30 and 50 RACEmethods

Wang et al., 2001

CsEI, Csv1, Csv2, Csv3 Toxins specific forNaþ-channels

Centruroides sculpturatusEwing

cDNA Corona et al., 2001

BmK IT3 Toxin Buthus martensii Karsch cDNA, RT-PCR Yu et al., 2002Twenty-three sequences

similar to Ergtoxin(ErgTx)

Block ERG Kþ-channels Centruroides (C.) elegans,C. exilicauda, C. gracilis,C. limpidus limpidus,C. noxius and C. sculpturatus

cDNAs Corona et al., 2002

LqqIT2 Insect depressant toxin Leiurus quinquestriatusquinquestriatus

cDNA, RT-PCR Zaki and Maruniak,2003

BmK ITa and BmK ITb Depressant insectneurotoxins

Buthus martensi Karsch cDNA Wang et al., 2003

Cll9 Neurodepressant toxin Centruroides limpiduslimpidus Karsch

cDNA Corona et al., 2003

BotIT2 Anti-insect toxin Buthus occitanus tunetanus RACE-PCR amplification(Rapid amplificationof cDNA ends)

Bel Haj Rhouma et al.,2003

BmTx3 Alpha-KTx Buthus martensi Karsch cDNA Huys et al., 2004BotIII Scorpion toxin Naþ Buthus occitanus tunetanus Genomic DNA Benkhadir et al., 2004Phaiodotoxin Insect-toxin Anuroctonus phaiodactylus cDNA Valdez-Cruz et al.,

2004aCOX I and COX II Cytochrome oxidase

I and IIC. sculpturatus cDNA Valdez-Cruz et al.,

2004b

(continued on next page)

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Table 1 (continued )

Peptide Funtion Scorpion species Template or/and method Reference

Thirteen genes andCOX I and COX II

Several toxins andcytochrome oxidase I and II

C. exilicauda cDNA Valdez-Cruz et al.,2004b

Lqh-dprIT(3) Anti-insect selectivedepressant toxin

Leiurus quinquestriatushebraeus

cDNA library, degeneratedprimer and specific primer

Strugatsky et al., 2005

BmKX Kþ toxin Buthus martensi Karsch cDNA and 30 and 50 RACEmethods

Wang et al., 2005

BmalphaTX14 Neurotoxin Buthus martensii Karsch cDNA Lü et al., 2005BmCa1 Calcium channel

toxin-like geneMesobuthus martensiiKarsch

Genomic DNA Zhijian et al., 2006a,b

BmK alphaIV Modulator of sodiumchannels

Buthus martensi Karsch Genomic DNA Chai et al., 2006

Heteroscorpine-1 (HS-1) Scorpine Heterometrus laoticus Genomic DNA Uawonggul et al., 2007Pha1, Pha2, Pha3, Pha4,

Pha5Phospholipases Anuroctonus phaiodactylus cDNA Valdez-Cruz et al., 2007

Ecdysone receptor andthe Retinoid X receptor

Heterodimeric receptorcomplex

Liocheles australasiae cDNA Nakagawa et al., 2007

BmKAGAP Antitumor-analgesic peptide Buthus martensii Karsch Genomic DNA Cui et al., 2010MeuTx3B a-KTx Scorpion Mesobuthus

eupeuscDNA and 30 and 50 RACEmethods

Gao et al., 2011

V. Quintero-Hernández et al. / Toxicon 58 (2011) 644–663648

MeuTx3 B from Mesobuthus eupeus (Gao et al., 2011), StCT1for Scorpiops tibetanus (Yuan et al., 2010), vejovine fromVaejovis mexicanus (Hernández-Aponte et al., 2011),NaScTxs from Rhopalurus junceus (García-Gómez et al.,2011), BmCa1 from Mesobuthus martensii Karsch (Zhijianet al., 2006b), NaScTxs from Androctonus crassicauda(Caliskan et al., 2006), scorpine from Pandinus imperator(Conde et al., 2000) and NaScTxs from C. noxius (Becerrilet al., 1993; Vazquez et al., 1995; Selisko et al., 1996).

3.2. Specific screening of ESTs from spider venom glands

Several cDNA libraries have been constructed from thevenom glands of spiders such as Phoneutria nigriventer(Penaforte et al., 2000; Cardoso et al., 2003) andMacrothelegigas (Satake et al., 2004)with the aim tofind specific spidertranscripts. Surprisingly, in addition of obtaining thespecificmRNA transcript, several other transcripts that codefor peptide isoforms have been found. Of those peptideisoforms several are either absent or in lowquantities in thecrude spider venom, implying that the spider venom glandproduces peptide toxins that are recycled or that aredifficultto fold within the cell glands. Other explanations to theseobservations could include: stress-dependent induction ofthe transcription/translation machinery, or the pharmaco-logical phenomenon of ‘synergy’ between 2 peptides(Wullschleger et al., 2005) in which a low quantity ofa neurotoxin can sufficiently and synergistically interactwith a neurotoxin (or vice versa), though the presence andactivity of proteases is not discarded. These peptides in lowquantities as well as the most representative peptide frac-tions have revealed high identities among families ofinsecticidal peptides isolated from spiders from differentgeographical areas. Examples of such peptide families comefrom the hexathelid spider Macrothele gigas from SouthJapan (Satake et al., 2004) and the theraphosid spidersOrnithoctonus huwena (Jiang et al., 2008) from China andBrachypelma smithi from Mexico (Corzo et al., 2008).

4. Random screening of ESTs

4.1. Random screening from scorpion venom glands

The first EST screening was performed by constructinga cDNA library from a single pair of venom glands of theMexican scorpionHadrurus gertschi. This pioneer analysis ofa non-Buthidae scorpion resulted in 147 high-quality ESTsthat allowed the authors to examine the molecular contentof the venom gland (Schwartz et al., 2007). Similar studieshave been conducted with Buthidae scorpions - Buthusoccitanus israelis (Kozminsky-Atias et al., 2008), Tityus dis-crepans (D’Suze et al., 2009), Lychas mucronatus (Ruiminget al., 2010), and Hottentotta judaicus (Morgenstern et al.,2011), and other non-Buthidae scorpions (Scorpiops jen-deki, Euscorpiidae, Ma et al., 2009; Opisthacanthus cayapo-rum, Liochelidae, Silva et al., 2009; Heterometrus petersii,Scorpionidae, Ma et al., 2010;) species, showing strikingdifferences regarding the diversity and abundance of toxin-like sequences.

Except for the one on H. judaicus (Morgenstern et al.,2011), these reports share a methodological principle: theRNA was extracted 2–5 days after venom extraction byelectric stimulation, whichmeans that the gland is engagedin regenerating its venom; this might be a useful conditionto explore the diversity of toxin transcripts that are notnecessarily actively transcribed once the venom has beenproduced. Indeed, the analysis of regenerating glandsrevealed an important enrichment of their transcriptomeswith toxin-like sequences, having from 50% up to 78% oftotal ESTs in Buthidae, and 30–44% in non-Buthidaespecies, classified as venom components (Table 2).Besides the previously characterized toxin types, (most ofthem neurotoxins and other ion channel toxins), some newvenommolecules such as lipolysis activating factors (LVPs),phospholipase A2 (PLA2), scorpion venom serine proteases(SPSV), metalloproteases, tick salivary protein homologs,cytolytic peptide precursors and other highly expressed

Table 2Classification of the transcripts (ESTs and clusters) obtained from differentscorpion species.

Species Family Sequences %EST Reference

Hadrurusgertschia

Caraboctonidae 147 ESTs68 clusters

31% VC19% CP50% U/H

Schwartzet al., 2007

Buthus occitanusisraelisa

Buthidae 420 ESTs 78% VC–

–

Kozminsky-Atias et al.,2008

Opisthacanthuscayaporuma

Liochelidae 118 ESTs61 clusters

36% VC39% CP21% U/H

Silvaet al., 2009

Tityusdiscrepansa

Buthidae 127 ESTs51 clusters

50% VC13% CP37% U/H

D’Suzeet al., 2009

Scorpiopsjendekia

Euscorpiidae 871 ESTs293 clusters

40% VC30% CP24% U/H

Ma et al.,2009

Lychasmucronatusa

Buthidae 738 ESTs380 clusters

55% VC22% CP23% U/H

Ruiminget al., 2010

Heterometruspetersiia

Scorpionidae 486 ESTs184 clusters

68% VC20% CP12% U/H

Maet al., 2010

Hottentotajudaicusb

Buthidae 537 ESTs283 clusters

24% VC39% CP37% U/H

Morgensternet al., 2011

Abbreviations: VC, venom component; CP, cellular process; U/H,unknown/hypothetical.

a Active or replenishing gland.b Resting or replete gland.

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cystein-rich proteins with signal peptides, were found atthe transcriptional level. It is worth mentioning that manytranscripts are so far those of putative protein precursors,as a high number of ESTs do not show significant similari-ties when compared to different databases. Proteomicstudies with scorpion venoms (reviewed in Rodríguez de laVega et al., 2010), have strengthened the conception thatthere is a remarkable amount of toxins to be described yet.Taken together, these observations suggest that there aretranscripts of unknown function that might represent newscorpion-specific gene families.

Table 3cDNA random transcripts obtained from different scorpion species.

Species Family # ESTs # clusters

NaScTx a-KTxs

Hadrurus gertschia Caraboctonidae 147 68 2.9Heterometrus

petersiiaScorpionidae 486 184 2.2

Opisthacanthuscayaporuma

Liochelidae 118 61 1.6

Scorpiops jendekia Euscorpiidae 871 293 2.4Tityus discrepansa Buthidae 127 51 27.4 11.8Lychas mucronatusa Buthidae 738 380 3.2 2.9Hottentotta judaicusb Buthidae 537 283 6.7 5,6Buthus occitanus

israelisa,cButhidae 420 75 52 25

a regenerating venom glands.b replete venom glands.c all open reading frames (ORFs) were inspected for the presence of a putative

As can be observed inTable 3, the number of cDNA clonesobtained in transcriptome studies vary from 118 to 871,resulting in 51–380 unique sequences, meaning that thenumber of unique sequences (or clusters) constitute around40–50% of the number of cDNA clones (or ESTs) analyzed.

Apart from new venom component precursors, alllibraries describe gene products related to cellularprocesses important for venom gland function, includinghigh protein synthesis, tuned post-translational processingand trafficking.

Table 3 shows the distribution of the unique sequenceswithin the diverse toxin classes. The resulting distribution isgreatly related to the scorpion family. Indeed, sodiumchannel modulator toxins (NaScTx) have been describedonly inButhidae scorpions,which include the scorpions thatare considered of medical importance. Human envenom-ization by scorpions is a commonmedical problem and fatalaccidents are commonly reported in many tropical andsubtropical countries, especially among children. Thephysiological manifestations of human envenoming areexplained by the ability of the NaScTxs to act on sodiumchannels on neuronal terminals, leading to the depolariza-tion of axonal membranes and the consequent release ofcatecholamines and acetylcholine which in turn stimulatevarious organs, including the gut, heart and vascular tissue(Couraudand Jover,1984; Freire-Maia andCampos,1989). InButhidae scorpion transcriptomes, NaScTx precursorsrepresent the most abundant venom content clusters.Otherwise, b-KTxs, scorpine-like, antimicrobial peptides(AMPs), and PLA2 are more abundant in non-Buthidaescorpions (Diego-García et al., 2007).

The transcriptome of L. mucronatus (Ruiming et al.,2010) revealed not only a high level of toxin-likesequences (56% of the total ESTs), but also importantdifferences of the transcriptional profile of two geograph-ically distinct populations (Yunnan and Hainan-sourced).For example, while long chain NaScTx and bKTx weremore abundant in one population (Hainan region), thetranscription of the a type of potassium channel toxins was2-fold higher and more diversified in primary structures in

% of total clusters

k-KTxs b-KTxsandscorpines

calcines AMPs BPPs PLA2 anionicpeptides

glicine-rich

1.5 2.9 1.5 1.51.6 1.1 0.5 2.7 1.1 1.1

3.3 1.6 8.2 1.6

0.7 0.3 3.4 0.32 2

0.3 0.3 0.5 0.3 0.3 0.30.3 0.7 0.31.3 1.3

leader sequence or a known cysteine-based pattern.

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the other group of scorpions (Yunnan region). This impliesthat the variability in proportion and diversity of venomcomponents within the same species might also depend onthe environmental conditions. In this case as well as for theS. jendeki transcriptome, several polymorphisms wereobserved in the toxin transcripts. Such differences can beattributed to variations in scorpion populations, since thecDNA libraries were constructed with RNA extracted frommore than 50 specimens.

In addition, a more recent study focused on the analysisof ESTs derived from “resting” or “replete” venom glands(Morgenstern et al., 2011). In this case, the cDNA library wasconstructed using one specimen of the Israeli Buthidaescorpion H. judaicus that had been starved for 14 days andhad not been stimulated to produce venom during thisperiod of time. Compared to other species, the transcrip-tional profile maintained a low level of toxin encodingtranscripts (even lower than non-buthid species), corre-sponding to 24% of the total ESTs, with some atypicalproportions of toxin types. Indeed, the abundance of NaScTxwas relatively low, whereas KTxs were the most abundanttype of transcripts, which is uncharacteristic of buthidspecies.Moreover, the proportionof the twomajor classes ofNaScTx showed that the a type was under-represented (theratio a:b was 24:76). It was also noticed that a significantfraction of the toxin-like transcripts were heavily mutatedand thus, were unlikely to be translated. Most of themcorrespond to non-depressant NaScTx, which had skippedstop codons, had abnormal accumulation of mutations orlacked the signal peptide sequences. Surprisingly, BjxtrIT,which is considered amajor component of the venomof thisscorpion (approximately1% of the dry weight of crudevenom)was not identified at the transcriptional level. Theseintriguing observations might be explained by the fact thatthe transcriptome does not directly reflect the venomcontent, but rather the transcript levels which are necessaryto maintain the venom gland in a “ready to act” state.

On the other hand, from the cDNA library constructedwith regenerating venom glands from Hadrurus gerstchi(Schwartz et al., 2007), the authors were not able to iden-tify the transcript for HgeTx1, one of the most abundanttoxins present in this venom (Schwartz et al., 2006) evenafter the use of specific primers for PCR amplificationdesigned based on the amino acid sequence of this toxin(unpublished data of our group).

Taken together, the transcriptional profiles of regener-ating and replete venom glands highlight our lack ofknowledge regarding the genomic organization of the toxinloci, the cellular processes and molecular dynamics thattake place in order to assemble, store and maintain suchcomplex protein mixtures. Even though new toxin-likesequences were derived with transcriptome approaches,their presence in the proteome of the gland as well as theiractivity will need to be validated.

4.2. Random screening of ESTs from spider venom glands

One of the first cDNA libraries, fromwhich cDNA cloneswere randomly sequenced was obtained from the wholebody of the spider Araneus ventricosus (Chung et al., 2001).From this cDNA library 385 clones were sequenced,

revealing that 63.6% and 37.4% of such sequences belong tocellular processes and to unknown proteins, respectively(Table 4). The fact that none of the clones corresponded toany already known toxin-like venom component demon-strates the unique specialization of a spider venom gland toproduce peptide toxin components (Table 4). After thiswork, similar studies were conducted using RNA extractedfrom the venomous glands of other spider speciesbelonging to the Agelenidae, Lycosidae, Sicariidae andTheraposidae families, for random screening of ESTs andspecific screening of toxin sequences. The abundance ofvenom components reported in these studies spans from20% to more than 80% of total ESTs (Table 4). For example,Koslov et al. (2005) compared two different approaches toobtain specific sequence information from an EST libraryconstructed from a pair of venom glands of the spiderAgelena orientalis. In one of the approaches, a specificstructural marker was introduced to find rich cysteinevenom peptides. The analysis of 150 polypeptides usingsuch specific structural marker resulted in 48 toxin-likestructures with ion channel inhibitor motifs, whichinclude several previously isolated toxins such as Ageleninfrom Agelena opulenta and 25 homologous sequences, 15homologous sequences of Agatoxin 2 and Agatoxin 3 withlow identity to Agatoxin-IIIA from the spider Agelenopsisaperta, and only 4 new primary structures from A. orientalis.Moreover, the most important finding was that using sucha specific structural marker permitted the identification oftoxin-like structures that overall exceed two thirds of thewhole database sequences.

Regarding clinically important spiders, it is known thatthe bite of spiders belonging to the genus Loxosceles canproduce severe clinical symptoms that, in some cases, maylead to human death. Among all the venom components ofLoxosceles spiders, sphingomyelinase D (SmaseD) is themost investigated and characterized toxin because of itsbiological effects. It causes dermonecrosis, acute renalfailure, massive inflammatory responses and hemolysis(Olvera et al., 2006). Given its medical relevance, the tran-scriptional profiles of the venom glands of two differentspecies of this genus, Loxosceles laeta and Loxosceles inter-media, have been recently described, showing strikingdifferences in terms of toxin abundance (Fernandes-Pedrosa et al., 2008; Gremski et al., 2010). The cDNAlibraries were constructed from RNA extracted from thevenom glands of 50 (L. laeta) and 350 (L. intermedia) spidersfive days after venom extraction. As described in previoussections, this procedure allows the glands to be activelyengaged in venom production. While SmaseDwas themostabundant component of the profile of L. laeta, accounting for16.3% of total ESTs and 43% of toxin-like clusters, it was farless represented in L. intermedia, where insecticidalpeptides such as LiTx accounted for almost 50% of the toxin-like encoding transcripts and 23%of total ESTs. In both cases,several toxin-like peptides and enzymes were identified.They include Astacin-like metalloproteases, serine prote-ases and neurotoxins such as Magi-3 from Japanese Mach-rotele gigas, hyaluronidases, lectins, lipases and venomallergens. These observations confirmed the rich source oftoxin-like peptides in brown spiders and their evolutionaryrelationship with spiders from different families.

Table 4cDNA random transcripts obtained from different spider species.

Species Family Sequences %EST Reference

Araneus ventricosusa Araneidae(Araneomorphae)

385 ESTs 0% VC63% CP37% U/H

Chung et al., 2001

Agelena orientalis Agelenidae(Araneomorphae)

150 ESTs37 contigs322 singletons

86% VC Kozlov et al., 2005

Loxoceles laeta Sicariidae(Araneomorphae)

3008 ESTs1357 clusters

31% VC33% CP25% U/H

Fernandes-Pedrosa et al.,2008

Haplopelma schmidti(Ornithoctonus huwena)

Theraposidae(Mygalomorphae)

468 ESTs24 contigs65 singletons

68% VC13% CP19% U/H

Jiang et al., 2008

Chilobrachys jingzhao Theraphosidae(Mygalomorphae)

788 ESTs85 contigs271 singlets

31% VC54% CP15% U/H

Chen et al., 2008

Loxoceles intermedia Sicariidae(Araneomorphae)

1843 ESTs538 clusters

43% VC10% CP47% U/H

Gremski et al., 2010

Haplopelma hainanum(Ornithoctonus hainana)

Theraposidae(Mygalomorphae)

1049 ESTs 20% VCb Xing et al., 2010

Citharischius crawshayi Theraposidae(Mygalomorphae)

236 ESTs14 contigs30 singletons

25% VC41% CP34% U/H

Diego-García et al., 2010

Lycosa singoriensis Lycosidae(Araneomorphae)

833 ESTs331 clusters

69% VC17% CP14% U/H

Zhang et al., 2010

VC, venom component; CP, cellular process; U/H, unknown/hypothetical.a cDNA from the whole body of the spider.b screening using specific toxin oligonucleotides.

V. Quintero-Hernández et al. / Toxicon 58 (2011) 644–663 651

Another interesting report concerns the EST analysis of20 venom-replenishing glands (removed 4 days aftervenom extraction) of the Chinese wolf spider Lycosa sin-goriensis. In this report, 69.1% of the total ESTs were foundto be similar to toxin-like sequences (Table 4), however,they showed an uncommon cystein pattern. Indeed, noneof the full-length toxin precursors seemed to form thetypical inhibitor cystine knot (ICK) motif since they show atleast eight Cys residues in their primary structures andthus, they could potentially form 4–6 disulfide bonds. Someof these sequences were similar to the antagonists ofvoltage-sensitive calcium channels, PNTx3-1, PNTx3-2 andu-agatoxin-4B, however, it is possible that many of thesetoxin-like peptides (if confirmed in the proteome)will havedifferent structural motifs and therefore, probably stillunknown biological effects.

Finally, the randomscreening of ESTs has been combinedwith proteomic data from the soluble venom of spiders.Recently, two interesting communications comparing thetranscriptome and venom analysis of the African spiderCitharischius crawshayi (Diego-García et al., 2010) and theChinese spider Haplopelma hainanum (Tang et al., 2010)were published. By means of combining protocols of tran-scriptomics, venomics, and biological assays, these authorsdetermined the molecular masses of most of the peptidesfrom the soluble venom and the ESTs from their venomglands. Sequence comparisons from 236 ESTs revealedinteresting and unique sequences, corresponding to toxin-like and other components from C. crawshayi. Moreover,mass spectrometry analysis of such venom fractionsshowed more than 600 components, which contrast withnearly 59 (25%) toxin-like ESTs previously found (Table 4). In

a similar way, nearly 420 peptide toxins were detected bymass spectrometry in the soluble venom of H. hainanum.However, using specific primers for toxin screening, 207peptide precursors were deduced from ESTs and 82 fromgenomic DNA. After redundancy removal, only 192 maturesequences were identified by the three approaches. Thepartial overlap between proteomic and transcriptomicresults might be explained by differential processing ofpeptides and pro-peptides, and more complex post-translational modifications that lead to a great number ofmasses identified in the venoms, which will not necessarilycorrelate with the toxin-like transcript abundance. There-fore, aswediscussed inprevious sections, the transcriptomeof a venom gland does not directly reflect the venomcontent.

Taken together, these studies show that the relativeabundance of toxin transcripts and toxin peptides repre-sent an important bias toward the characterization of themost abundant venom components depending on themethodological approach used. It is possible to foresee thatcomplex post-translational modifications, gene duplicationand local hypermutation may be responsible for the largemolecular diversity of spider toxins within the solublevenom, and that this diversity has to be elucidated bycombining different experimental strategies, such asgenomics, transcriptomics, proteomics and biologicalassays.

5. High throughput screening of arachnid ESTs

The recent development of massive sequencing proto-cols offers an excellent opportunity to explore the

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transcriptional universe of venomous animals, coveringlow abundance transcripts that are difficult to incorporatein traditional cDNA libraries. The African scorpion P. impe-rator was the first species to be examined under thisapproach (Roeding et al., 2009). One single 454-FLX pyro-sequencing run produced around 429,000 reads(223 � 50 bp long), that were assembled into 8334 contigs.The quality of the assembly was assessed by analyzinghemocyanin sequences (eight hemocyanin subunits wereidentified, six of whichwere complete). Homology searchesrevealed that around 72% of the sequences shared signifi-cant identity with known proteins; the most common GeneOntology terms corresponded to cellular, metabolic andmulticellular organism processes. In contrast to the above-mentioned reports, this cDNA library included transcriptsfrom thewhole scorpion body and telson, a fact that did notallow determining the specificity of gene expression. Thisstudy did not focus on the discovery of new toxins, butrather aimed at making a comprehensive multigene-basedphylogenetic study that included 149 orthologous genesfrom 67 different taxa, in order to understand arthropodrelationships with the inclusion of the P. imperator data.

Being non-model organisms, the catalog of scorpionsequences in public databases is very limited and highlybiased for toxin peptides and transcripts. Therefore, itwould be interesting to apply high throughput sequencingplatforms to make qualitative and quantitative compari-sons between gland-specific ESTs and body transcripts, inorder to improve our understanding of important biologicalaspects of these arthropods.

The development of high throughput sequencingprotocols could offer an opportunity to explore the lowabundant transcripts such as short antimicrobial and toxinpeptides in spider venoms that are generally difficult to berevealed by means of traditional cDNA libraries. Until now,no reports of massive sequencing of spider venom tran-scripts can be found in the literature.

6. Cell systems for heterologous expression of spiderand scorpion peptides

Spider and scorpion toxins are interesting tools for thestudy of cell receptors. They are short peptides usuallycontaining several disulfide bridges. Three main ways forobtaining such priced ligands are from natural sources,chemical synthesis or heterologous expression. The naturalway for obtaining spider or scorpion toxins requires fieldcollection or laboratory rearing of specimens for obtainingtheir crude venoms. The lack of living specimens for venomcollection, the high price and small catalog of lyophilizedvenoms available from providers, and moreover, theseveral steps of chromatography, represent bottlenecksthat limit the available quantities of venom components.When affordable, peptide chemical synthesis has been anoption to avoid purification from natural sources. It has,however, several limitations: the high cost, the time itconsumes, the technical challenge associated with thesynthesis of long peptides, and the need for in vitro foldingsteps for cysteine-rich peptides to be active, which reducesthe yield of the chemically synthesized peptides (Lecomteet al., 1998).

Therefore, genetic engineering has becomean importanttool to produce large amounts of the spider and scorpionpeptides of interest with a relative low cost and the possi-bility of producing not only the wild-type peptide but alsovariants using site-directedmutagenesis. These approachesmake possible to obtain enough material to elucidate themechanism of action of the peptides and their structure.

Basic to the successful production of recombinant spiderand scorpion toxins is to choose a system that permits thecorrect folding of the peptide, with precise disulfide bridgesformation, assuring in this way the effectiveness of thebiological activity (Estrada et al., 2007; Escoubas et al.,2003). Hence it is necessary to evaluate and consider thefeasibility of the different expression systems. For example,most bacterial systems are not able to conduct post-translational modifications on proteins, thus, some of thebacterial expression systems could be inappropriate for theproduction of active heterologous proteins. Additionally,in vitro modifications may be required to obtain functionalpeptides. In the following sections, several expressionsystems in bacteria, yeasts and eukaryotic cells of spiderand scorpion venom peptides will be presented. Eachsystem has its own advantages and disadvantages. We willfurther discuss their characteristics and accomplishments.

6.1. Bacteria

To date, almost half of all the expressed scorpion(Table 5) and spider (Table 6) peptides have used Escherichiacoli as the expression host. This is therefore themost widelyused system for this task. It is technically accessible, fast,and cost-effective, with simple culture conditions, easilyscalable, and E. coli is an easily manageable organism forwhichmany strains and vectors are available aswell as fine-tuned protocols for genetic manipulation (Terpe, 2006).

Several disadvantages are also noteworthy. E. coli cyto-plasmic proteins do not generally contain disulfide bondsand several pathways exist in order tokeep those proteins inthe reduced form (Stewart et al., 1998). This fact playsagainst the correct folding of cysteine-rich bioactivepeptides, at least those expressed in the cytoplasm (seebelow). On the other hand, the codon usage of bacteria andarthropods is different. This is sometimes reflected in lowyields and/or the production of truncated species. Theconsequence is that, sometimes, the cloned codingsequence, amplified from the natural source, cannot bedirectly used for expression and a new gene with the pref-erential codon usage for E. coli has to be assembled byrecursive PCR or by chemical synthesis.

6.1.1. Scorpion peptidesSeveral successful cases of scorpion peptide expression

in E. coli systems have been reported. They are mainly thoseof ion channel-modulating and analgesic peptides (Table5). The most widely used strains are BL21 and its deriva-tive BL21(DE3), this last being the one of choice for the T7promoter-driven expression vectors of the pET family(Novagen). The expression of the most potent toxins fromscorpions Parabuthus granulatus and Centruroides suffusussuffusus (Pg8 and CssII, respectively) are two examples ofsuccessful expression of recombinant toxins employing

Table 5Plasmids and cell systems for scorpion peptide expression.

Vector Toxin or Peptide Scorpion species Yield Reference

BacteriaEscherichia coli

Bl21 DE3pSR9 Charybdotoxin (CTX), a Kþ

channel blockerLeiurus quinquestriatus 30 mg/L Park et al., 1991

Escherichia coli pIN III(Ippp�5)A2 andpIN III ompA2

Insectotoxin I5A Buthus eupeus Trace amount Pang et al., 1992

Escherichia coli PCSP 105 NTX, noxiustoxin, a Kþchannel-blocking peptide

Centruroides noxiusHoffmann

1.3 mg/L Martínez et al., 1996

Escherichia coliBL21 (DE3)

PET-11 cK Anti-insect selective scorpiondepressant neurotoxin LqhIT2

Leiurus quinquestriatushebraeus

500 mg/L Turkov et al., 1997

E.coli, a thioredoxinmutant hostbacterial cell,AD 494(DE3)pLysS

pET15b. Beta-neurotoxin Css II C. suffusus suffusus 1–2 mg/L Johnson et al., 2000

E. coli BL21 (DE3) PET28a Anti-neuroexcitation peptide(ANEP)

Buthus martensii Karsch 1.5 mg/L Zhang et al., 2001

E. coli BL21 (DE3) PET-28a BmK IT3 Buthus martensii Karsch Nr Yu et al., 2002Escherichia coli BL21 pGEX-5x-1

(GST fusion protein)BmKIM toxic to both mammaland insects.

Buthus martensii Karsch 1–2 mg/L Peng et al., 2002

Escherichia coliBL21 (DE3)

PET-28a Antitumor-analgesic peptide(AGAP)

Buthus martensii Karsch 2.5 mg/L Liu et al., 2003a

E. coli BL21 (DE3) PGEX-5X-1(GST fusion protein)

BmTXKbeta Buthus martensii Karsch 2 mg/L Cao et al., 2003

Escherichia coli CMK pMalC crustacean toxin, Cn5 Centruroides noxiusHoffmann

5 mg/L Garcia et al., 2003

Escherichia coli HB101 pEZZ-18 (fusionprotein with theZZ domain fromStaphylococcalprotein A)

Alpha-scorpion toxin BotIIIthe most toxic protein

Buthus occitanus tunetanus 2 mg/L Benkhadir et al., 2004

E. coli BL21 (DE3) PExSecI (systemIgG bindingdomain-ZZ ofProtein A is fusedto BmK Mm2)

Neurotoxin BmK Mm2 Buthus martensii Karsch 1.6 mg/L Fu et al., 2004

Escherichia coli pMALp AaH II fusedwith MBP

Na þ toxin, AaH II Androctonus australisHector

0.3 mg/L Legros et al., 2005

E. coli BL21 (DE3) PExSecI (The IgG-binding domain-ZZ of protein A isfused to rBmK Cta)

Chlorotoxin-like peptiderBmK Cta

Buthus martensii Karsch 7.8 mg/L Fu et al., 2005

Escherichia coli PET3a BmK IT-AP, an excitatoryinsect toxin

Buthus martensii Karsch 0.5 mg/L Li et al., 2005

E. coli strain[AD494(DE3) pLysS]

PET15b (poly-histidine-taggedfusion protein)

Neurotoxin LqqV Leiurus quinquestriatusquinquestriatus

1.5 mg/L Banerjee et al., 2006

E. coli BL21 (DE3) pET-28a(þ) BmK aIV Buthus martensii Karsch 2 mg/L Chai et al., 2006Escherichia coli

BL21 trxB (DE3)PET32a Analgesic peptide BmK

AngM1Buthus martensii Karsch 25 mg/L Cao et al., 2007

E. coli BL21 PQE30 Beta neurotoxin CssII Centruroides suffusussuffusus

24.6 mg/L Estrada et al., 2007

E. coli BL21 (DE3) pTWIN1 Insect toxin (BmK IT) Buthus martensii Karsch Nr Xu et al., 2007Escherichia coli BL21

and OrigamiPQE30 Pg8 Parabuthus granulatus 5 mg/L García-Gómez et al.,

2009Escherichia coli TG1 pSyn1 ErgTx1 Centruroides noxius

Hoffmann6–10 mg/L Jimenez-Vargas et al.,

2011E. coli BL21 (lDE3) pSYPU BmK AGP-SYPU1 analgesic

peptideButhus martensii Karsch Nr Wang et al., 2011

Escherichia coliBL21(DE3)

pTWIN1 Insect neurotoxin (BmK IT) Buthus martensii Karsch 3 mg/0.5 L Fu et al., 2011

YeastsSaccharomyces

cerevisiaePaC2 Insectotoxin I5A Buthus eupeus 0.05–0.1 mg/ml Pang et al., 1992

Yeast PMA 91 Insect toxin 1 (AaH IT1) Androctonus australisHector

4 mg/L Martin-Eauclaireet al., 1994

Saccharomycescerevisiae

pVT102U/a BmK M1 neurotoxin Buthus martensii Karsch 5–10 mg/L Shao et al., 1999

Yeast secretionsystem

pVT102U/a BmP05 Ca þ toxin Buthus martensi Karsch 8 mg/L Wu et al., 2002

Yeast pPIC9K Neurotoxin BmalphaTX14 Buthus martensii Karsch 120 mg/L Lü et al., 2005

(continued on next page)

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Table 5 (continued )

Vector Toxin or Peptide Scorpion species Yield Reference

Pichia pastoris pGAPZaB (ButaITfused N-terminallyto a GNA polypeptide)

Lepidopteran-selectivetoxin ButaIT

Mesobuthus tamulus 25–35 mg/L Pham et al., 2006

Pichia pastoris X-33 pPICZalpha-A Synthetic antimicrobialpeptide (CP)

N/A 271 mg/L Niu et al., 2008

Pichia pastoris PPIC9K Insect neurotoxin LqhIT2 Leiurus quinquestriatushebraeus

9 mg/L Li and Xia, 2008

Pichia pastoris Nr Analgesic peptide BmKAngM1

Buthus martensii Karsch 500 mg/L Yang et al., 2009

Pichia pastoris pGAPZaB (ButaITfused to a GNA)

Insect toxin ButaIT Mesobuthus tamulus 25 mg/L Fitches et al., 2010

Insect and animalcells

Monkey kidneyCOS-7 cells

PCDV-1 Neurotoxin toxin II Androctonus australis 0.2 mg/106 cells Bougis et al., 1989

NIH/3T3 mousefibroblast cells

Vector with thetranscriptionalcontrol of a murineretro-viral longterminal repeat.

Insecticidal neurotoxin(AaIT)

Androctonus australis Nr Dee et al., 1990

Tn-5B1-4 cells pFastBacHTcbaculovirus transfervector

Insect-specific neurotoxinBmK ITa1

Buthus martensii Karsch 20 mg/106 cells Liu et al., 2003b

HEK 293T cells pEGFP-N1 BmKK2 Buthus martensii Karsch Nr Zhijian et al.,2006a,b

HEK 293T cells pEGFP-N1 (eukayoticexpression vectorencoding GFP)

BmKK2 Buthus martensii Karsch Nr Dai et al., 2007

BaculovirusAutographa californica

nuclear polyhedrosisvirus (AcMNPV)

N/A Insectotoxin-1 Buthus eupeus N/A Carbonell et al., 1988

AcMNPV pAcUW2B Insect-specific neurotoxin Androctonus australisHector

400 ng/ml Stewart et al., 1991

Double subgenomicSindbis (dsSIN) virus

N/A Insect-specific toxin(AaHIT)

Androctonus australisHector

N/A Higgs et al., 1995

Trichoplusia ni nuclearpolyhedrosis virus(TnNPV)

Transfer vectorpSXIV VI þ X3

Insect-specific neurotoxinAaIT

Androctonus australis N/A Yao et al., 1996b

Baculovirus BmNPV BmM14/BmAaIT AaIT, an insect-selectiveneurotoxic peptide

Androctonus australisHector

N/A Elazar et al., 2001

AcMNPV pAcUW21 Buthus tamulus insectselective toxin (ButaIT)ButaIT-NPV

Mesobuthus tamulus N/A Rajendra et al., 2006

AcMNPV A polyhedrin-positiverecombinantAcMNPVvAcP(hsp70)EGFP/P(pag90)IT(2)

LqhIT(2), an insect-specificneurotoxin fusion withgreen fluorescenceprotein (EGFP

Leiurus quinquestriatushebraeus

N/A Jinn et al., 2006

Bac-to-Bac baculovirusexpression system

pFP Insecticidal scorpionneurotoxin AaIT

Androctonus australis N/A Regev et al., 2006

Densovirus (PfDNV) BmKIT1-GFP fusionprotein

Insect-specific toxinBmKIT1

Buthus martensi Karsch N/A Jiang et al., 2007

Transgenic plantsTobacco pMON316 Insectotoxin I5A Buthus eupeus N/A Pang et al., 1992Tobacco NC89 pNGY-2 Neurotoxin AaIT Androctonus australis N/A Yao et al., 1996aTransgenic tobacco

and tomato.PBI121 Analgesic-antitumor

peptide (AGAP)Buthus martensi Karsch N/A Lai et al., 2009

N/A not applicable Nr no reported.

V. Quintero-Hernández et al. / Toxicon 58 (2011) 644–663654

E. coli BL21 (García-Gómez et al., 2009 and Estrada et al.,2007). In both cases the vector of choice was pQE30 (QIA-GEN) and the genes for the toxins were fused to a His-tagcoding sequence for convenience, since it allowed rapidpurification of the product by Ni-NTA agarose column. Theexpressions were cytoplasmic and the products accumu-lated as exclusion bodies. The yields were high, but thenin vitro refolding procedures were required in order toobtain the active peptides.

As mentioned above, the E. coli cytoplasm does notnormally fulfill the requirements for the correct formationof disulfide bonds necessary for cysteine-rich scorpionpeptides to be active. Nonetheless, some E. coli strains havebeen engineered and the genes encoding for theThioredoxin-oxidase and Glutation-reductase have beenmutated, so that those strains now have an oxidizing cyto-plasm (Prinz et al., 1997). The CssII toxinwas also expressedin amutant E. coli strainwith oxidizing cytoplasm. The yield

Table 6Plasmids and cell systems for spider peptide expression.

Vector Tx Specie Yield Reference

BacteriaE. coli DH5 pGEX-KT Huwentoxin-1 Selenocosmia huwena 12.5 mg/L Li et al., 2000E. coli BL21(DE3) pGEX-2T J-ACTXHv1c Hadronyche versuta Nr Maggio and King, 2002E. coli BL21(DE3) pMAL-c2 PnTx-3-1 Phoneutria nigriventer 16 mg/L Carneiro et al., 2003E. coli BL21(DE3) pGEM/pET32a GsMTx4 Grammostola spatulata 0.3 mg/L Ostrow et al., 2003E. coli BL21(DE3) pGEX-2T u-Atracotoxin-

Hv1aHadronyche versuta Nr Tedford et al., 2004

E. coli(AD494(DE3)pLysS)

pET-32c Tx1 Phoneutria nigriventer Nr Diniz et al., 2006

E. coli BL21(DE3) pMAL ProTx-II Thrixopelma pruriens Nr Smith et al., 2007E. coli BL21(DE3) pET-32b Latarcin 2a Lachesana tarabaevi 3.2 mg/L Shlyapnikov et al., 2008YeastsPichia pastoris pGAPZaA SFI1 Segestria florentina 0.5–5 mg/L Fitches et al., 2004Pichia pastoris pPICZa GsMTx4 Grammostola spatulata 100 mg/L Park et al., 2008Saccharomyces

cerevisiaepYES2-DEST52 Lycotoxin 1 Lycosa carolinensis Nr Hughes et al., 2008

Saccharomycescerevisiae

pVT102U/a Lectin-like peptides Ornithoctonus huwena 2 mg/L Jiang et al., 2009

Saccharomycescerevisiae

pVT102U/a Jingzhaotoxin-34 Chilobrachys jingzhao. 4 mg/L Chen et al., 2009

Animal CellsSpodoptera

frugiperdaSf21

pAcLTX, pAcLITand pAcLMWP

a–latrotoxinaa -latroinsectotoxina8 kDa protein

Latrodectus mactans 2 pg/106 cells2 pg/106 cells10 pg/106 cells

Kiyatkin et al., 1995

Spodopterafrugiperda

Sf9/High-five

pFastBac a-latrotoxina Latrodectus mactans Nr Ichtchenko et al., 1998

DrosophilaS2 cells

pGEM PcTx1 Psalmopoeus cambridgei 0.4 mg/L Escoubas et al., 2003

Spodopterafrugiperda

Sf9/High-five

pFastBac Huwentoxin-1 Selenocosmia huwena 0.9 mg/L Ji et al., 2005

Transgenic plantsNicotiana tabacum J-ACTXHv1c Hadronyche versuta Nr Khan et al., 2006Nicotiana tabacum pBin19 Magi6 Macrothele gigas 17–22 mg/g Hernandez-Campuzano

et al., 2009

Nr-no reported.

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was low, but the peptide recovered from the soluble fractionwas biologically active (Johnson et al., 2000).

Yet another strategy to obtain the desired peptide withthe correct folding is to direct its expression to the peri-plasmwith the help of secretory signal peptides. In contrastto the cytoplasm, the E. coli periplasm is normally oxidizing,which favors the formation of the disulfide bonds. Theperiplasm however, is much smaller in volume comparedto the cytoplasm, so the amount of proteins produced inthis space is limited. Nonetheless, good yields have beenreported with this strategy. For example, toxin ErgTx1 fromthe scorpion C. noxius was expressed as a fusion proteinwith Thioredoxin and directed to the periplasm (Jimenez-Vargas et al., 2011). The combination of the carrierprotein and the oxidizing environment of the periplasmresulted in very good yields of the functional toxin withoutthe need for any refolding procedure.

6.1.2. Spider peptidesIn order to study spider venom peptide mechanisms it is

imperative to produce high amounts of active isoforms. Oneof the best expression systems studied to produce spiderpeptides is the use E. coli as the host cell. It is used both inthe cloning steps as well as for the expression of theproteins. Inmost cases, the nucleotide sequence that encode

the mature spider toxin is obtained from the laboratory dataor from public databases; in the later case, the gene could bechemically synthesized de novo and inserted into a cloningplasmid such as pBluesript II SK (þ) (Stratagene)(Hernandez-Campuzano et al., 2009), pGEM (Promega)(Escoubas et al., 2003), and others. The constructed gene isthen subcloned into expression plasmids like pET-32 (þ)(Stratagene) (Shlyapnikov et al., 2008). For most cases theproduct is expressed as a hybrid, that is, the spider gene isfused to a carrier protein. Examples of recombinantproduction of peptide toxins in E. coli followed by in vitrodisulfide formation include Huwentoxin I from the spiderSelenocosmia huwena (Li et al., 2000), and the J-Atracotoxinsfrom the spider Hadronyche versuta (Maggio and King,2002). Although production yields can be up to 10–20 mg/L (Li et al., 2000), the refolding step is usually the limitingstep to obtain sufficient amounts of active toxin, as therefolding yield can be less than 10% of the original product.Other strategies based on fusionproteins have permitted theproduction of soluble, folded forms of the toxins in E. coliusing a thioredoxin reductase-deficient strain (Ostrow et al.,2003) or glutathione-S-transferase (GST) gene fusionsystems (Tedford et al., 2004). Several drawbacks of themethodology include the need for protease or chemicalcleavage of the fusion proteins, lowering final yields and

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sometimes cleaving the toxin sequence itself, as well as thedifficulty in obtaining post-translational modifications,often necessary for toxin activity.

6.2. Yeasts

Pichia (P.) pastoris is the yeast host most commonly usedfor heterologous expression. It offers several advantagesover other yeast systems as Saccharomyces cerevisiae. Forexample, Pichia pastoris can be grown in methanol as theonly carbon source, at concentrations that would killalmost any other microorganism. It also grows to very highcell densities, which means higher protein yields perculture. Although not that simple to manipulate andmaintain as E. coli, nor as fast in protein production,P. pastoris has a very relevant advantage over the former: it iscapable of forming disulfide bonds (and other post-trans-lational modifications, as glycosilation) and correctly foldsthe cysteine-rich peptides (Cereghino and Cregg, 2000).

The sequence to be expressed in P. pastoris is usuallyplaced under the control of the alcohol oxidase 1 genepromoter, AOX1 (which is a strong promoter inducible bythe addition of methanol) and is fused to the secretorysignal peptide of the a-mating factor from S. cerevisiae, sothat the expressed protein is secreted to the growthmedium from where it is easily purified.

6.2.1. Scorpion peptidesExamples of scorpion venom peptides successfully

expressed in this system include analgesic peptides likeBmK AngM1 from Buthus martensii Karsch (Yang et al.,2009), insect-specific neurotoxins like LqHIT2 from Leiu-rus quinquestriatus hebraeus (Li and Xia, 2008) and anti-microbial peptides like the engineered recombinantpeptide CP (Niu et al., 2008). High yields (ranging from 9 to500 mg/L) of correctly folded and active peptides wereobtained in all cases.

6.2.2. Spider peptidesRecombinant spider peptides such GsMTx4 have been

expressed in P. pastoris (Invitrogen, Carlsbad, CA) using thepPICZaB vector containing the a-mating factor prepro-leader sequence (aMF signal sequence) which facilitatesthe expression of the recombinant protein; therefore theN-terminus of the spider peptide is fused to aMF factor andintroduced to the host yeast strain, P. pastoris GS115 byelectroporation. The pPICZaB vector is integrated into theyeast genome. Colonies are selected using minimal mediacontaining dextrose andhistidine ormethanol andhistidine.The P. pastoris GS115 host strain has a mutation in the histi-dinol dehydrogenase gene that prevents it fromsynthesizinghistidine. Furthermore, this vector allows alcohol to be usedas a nutritional source because it contains alcohol oxidaseactivity. Thus, colonies that expressed the recombinantprotein, growonaminimalmediumcontaininghistidineandmethanol (Park et al., 2008). Moreover, a snowdrop lectinfrom Galanthus nivalis agglutinin (GNA) was fused to aninsecticidal spider neurotoxin from the venom of Segestriaflorentina. The fusionproteinwas expressed also in P. pastorisusing the vector pGAPZaA (Invitrogen), and it was tested fortoxicity against the larvae of tomato moth Lacanobia

oleracea, the rice brownplant hopperNilaparvata lugens andthe peach-potato aphidMyzus persicae by incorporation intoartificial diets. The survival of the insects was significantlyreducedwhen fedon the fusionprotein. The abilityofGNA toact as a carrier protein of spider peptides to thehaemolymphof these insects, following oral ingestion, was confirmed bywestern blot when haemolymph taken from the insectscontained the GNA-immunoreactive proteins with the cor-responding molecular weights to GNA and to the fusionproteins (Down et al., 2006; Fitches et al., 2004). Severalother spider peptides such as Lycotoxin, Lectin-like peptidesand Gzhaotoxin-34 spider toxin have been expressedsuccessfully in yeast (Hughes et al., 2008; Jiang et al., 2009;Chen et al., 2009).

6.3. Animal cells and baculoviruses

Animal cells and baculovirus expression has also beenassayed for expression of scorpion and spider venomcomponents.

6.3.1. Scorpion peptidesThe BmKITa1 toxin from B. martensii Karsch was co-

expressed with the rat C-terminal amidating enzymepeptidylglycine a-amidating monooxygenase (PAM) incultured insect cells. C-terminal a-amidation is reported tobe a very important post-translational modification for theactivity of several neuropeptides. The co-expression resul-ted in the biologically active neurotoxin (Liu et al., 2003b).

The murine fibroblast NHI/373 cell line has been usedfor the expression of scorpion toxins, as is the case of theinsect-specific AaIT toxin from A. australis Hector (Deeet al., 1990). The produced AaIT was selectively toxic, justas the native toxin.

Toxin AaHII from A. australis Hector was one of the firstheterologously expressed toxins. COS-7 monkey kidneycells were used for expression and the recombinant proteinshowed the same biological and immunological activitiesas the native toxin (Bougis et al., 1989).

Although baculoviruses are commonly used as expres-sion vectors in conjunction with cultured insect cells toproduce recombinant proteins, there is another field ofapplications to the baculoviral systems that deserves specialconsideration. Since each baculovirus species hasa restricted range of invertebrate hosts that it can infect(usually closely related insect species) and due to the factthat they are not capable of infecting mammals or plantsand are therefore considered safe for humans, baculovirusesexpressing insect-specific scorpion toxins havebeenused asmodels for insect pest control. Examples are baculovirusesexpressing the AaIT toxin from A. australis Hector (Regevet al., 2006), the LqHIT2 toxin from L. quinquestriatushebraeus (Jinn et al., 2006) and Insectotoxin I from Buthuseupeus (Carbonell et al., 1988). All the recombinant baculo-viruses showed an increased insecticidal activity.

6.3.2. Spider peptidesThe firsts expressed spider toxins in insect cells were

the a-latrotoxins from L. mactans venom. They wereexpressed in insect cells from Spodoptera (S.) frugirpedausing a baculovirus expression system. Kiyatkin et al.

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(1995) used the derivative of the Autographa californicanuclear polyhedrosis virus (AcNPV) encoding the E. coli lacZgene in place of the wild-type polyhedrin gene codingsequences (AcNPV.lacZ), which was propagated in S. fru-girperda (Sf) cells. Then, the recombinant baculoviruses,carrying the full-length a-latrotoxins, were prepared by theco-transfection of Sf cells with the linearised AcNPV.lacZDNA and the transfer vectors. Finally, the recombinantviruses were isolated by plaque assay. The SDS/PAGEanalysis of their recombinant virus-infected cells revealednovel proteins that migratedwith sizes similar to the nativeneurotoxins from the spider venom. The identities of thea-latrotoxins were confirmed by immunoblot analysis(Kiyatkin et al., 1995; Ichtchenko, 1998; Volynski et al.,1999). Following this work, several short and long spidertoxins have been expressed in similar animal cells (Ji et al.,2005; Escoubas and Rash, 2004). For example, the shortpeptide toxin Psalmotoxin 1 (PcTx1), a potent and specificblocker of the ASIC1a proton-sensing channel, wasexpressed in the Drosophila melanogaster S2 cell expressionsystem. The final yield of the recombinant toxin was0.48 mg/L and it was identical in all aspects to the nativepeptide, and its three-dimensional structure in solutionwas determined (Escoubas and Rash, 2004). The syntheticgene was fused to the 22 amino acid signal peptide ofmelittin, a highly expressed bee venom peptide, to allowfor the efficient secretion of PcTx1 into the culture medium.The DNA fragment was ligated into a pGEM-T easy vector(Promega). After sequencing, the DNA construct wassubcloned into the Drosophila expression vector pMT/V5-His (InvitroGen) to allow for the recombinant expressionof PcTx1 in S2 cells of the Drosophila expression system(Escoubas and Rash, 2004).

Special attention requires the recombinant expressionof Sphingomyelinase-D and a-Latrotoxin the major toxiccompounds of the envenomization symptoms caused bythe brown spider Loxoceles sp. and the black widowspider Lactrodectus sp., respectively. Antibodies raisedagainst the recombinant Sphingomyelinase-D or a-Latro-toxin were capable of neutralizing the intoxicationsymptoms produced in mice by the venom of the spidersLoxoceles reclusa or Lactrodectus mactans, respectively(Olvera et al., 2006, 2007; Bugli et al., 2008). Additionally,the recombinant Sphingomyelinase-D is actually used asan antigen for the commercial production of F(ab0)2antibodies, the first commercial antivenon against thebite of Loxoceles spp obtained from a recombinant spidervenom toxin (Trade mark LOXMYN, COFEPRIS number020M2009SSA, Mexico).

6.4. Plant cells

Finally, plant cells have been used for expression ofscorpion and spider venom components.

6.4.1. Scorpion peptidesFew reports regarding the expression of scorpion toxins

in plants have been published. The first report of a trans-genic plant expressing a scorpion peptide was that oftobacco plants expressing Insectoxin 15A from Buthuseupeus (Pang et al.,1992). The gene coding for the analgesic-

antitumoral peptide AGAP from B. martensii Karsch wasintegrated into the plant genomic DNA in tobacco andtomato and was successfully expressed (Lai et al., 2009).Neurotoxin AaIT from A. australisHector was also expressedin NC89 tobacco plants resulting in transgenic plants withremarkable resistance to insect attacks (Yao et al., 1996a).

6.4.2. Spider peptidesTo investigate the in vivo function of spider toxins

expressed in plants, a 275-bp fragment that codes for themature spider neurotoxin Magi 6 cDNA without the leadersequence was cloned in a pBluescriptKS-derived plasmid(pJLU27) containing the tobacco mosaic virus 50-end leadersequence. The XbaI-KpnI insert was excised and subclonedinto the pBin19 vector, containing the 0.8 kb 35S-promoterand 0.3-kb NOS polyadenylation site. The construct wasintroduced by electroporation in Agrobacterium tumefa-ciens LBA4404 strain and used to transform Nicotianatabacum. Seventy-one fully regenerated and kanamycin-resistant tobacco plants were obtained after trans-formation with the Agrobacterium tumefaciens system.Twelve selected 35S::Magi6 lines expressed the transgeneand nine of them expressed from moderate to high levelsof Magi6 demonstrating that those transgenic plants wereresistant to insect attack (Hernandez-Campuzano et al.,2009). They also showed that the Magi 6 peptide accu-mulated in leaves at 4–6% of soluble protein that isequivalent to 17–22 mg/g of plant tissue, and this wassufficient to make plants resistant to insects. Comparisonof Magi 6 accumulation in transgenic tobacco to theexpression level of other bioinsecticide peptides revealsthat this is one of highest yield levels so far reached (Table6). Also, the peptide toxin u-ACTX-Hv1a (Hvt), from theHadronyche versuta spider, which functions as an antago-nist of calcium ion channels, was expressed in tobaccoplants and shown to confer insect resistance (Khan et al.,2006). However, an important question that remainsunanswered is how insecticidal spider toxins expressed intobacco plants affect insects, since oral administration ofactive insect neuropeptides such as Magi 6 is unlikely to besuccessful due to the insect gut enzymes that will probablydegrade them. However, the delivery of small peptidescould be obtained by means of protein carriers such aslectins.

7. Fusion proteins in heterologous expressed scorpionand spider toxins

Since most peptides heterologously expressed fromcloned genes of scorpions and spiders are relatively shortpeptides, the strategy frequently used is the cloning of thepeptide-coding sequence in framewith the gene of a carrierprotein (e. g. Thioredoxin, IgG-binding domain ZZ ofprotein A, GST), thus a recombinant fusion protein isproduced. Between the carrier protein and the peptide,a protease recognition site is conveniently placed bydesign, so that after expression, the peptide is cleaved freefrom the carrier. Examples of proteases used for thispurpose include Factor Xa and Enterokinase.

Pioneer works in this regard were performed withpeptides BmKIM and BmTXKb from the scorpion Buthus

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martensii Karsch (Peng et al., 2002 and Cao et al., 2003) andwith toxin Cn5 from Centruroides noxius (Garcia et al.,2003). BmKIM and BmTXKb were expressed as fusionproteins with GST and the recombinant products werethereafter digested with enterokinase and the activeBmKIM and BmTXKb were obtained with yields of 2 mg/L,without the need for any in vitro refolding. The Cn5 toxinfrom C. noxius was expressed in the cytoplasm of E. coli asa fusion protein with MalE, resulting in a yield of 50 mg/L.The free Cn5 was obtained after cleavagewith factor Xa andthe final yield of pure peptide (before folding) was in theorder of 5 mg/L.

The AaH II toxin of A. australis was expressed usinga similar strategy to the one mentioned above: the AaH IIgene was cloned into pMalp vector and expressed asa soluble protein fused to MBP (maltose-binding protein)that was transported to the E. coli periplasm. Althoughsatisfactory amounts of the fusion protein were produced,the recovery of the free recombinant toxin AaH II waspoor after cleavage with enterokinase (Legros et al.,2005).

Another successful example is the expression of thechlorotoxin-like BmK CTa peptide from the scorpionButhus martensii Karsch, this peptide was expressed in E.coli BL21 (DE3) using a pExSecI expression system inwhich the IgG-binding domain-ZZ of protein A is fused tothe N-terminal of rBmK CTa. The fusion protein, ZZrBmKCTa, was expressed in soluble form with a yield of 7.8 mg/L of culture. The domain-ZZ of fusion protein ZZrBmK CTawas removed by cleavage of an Asn–Gly peptide bondwith hydroxylamine. The rBmK CTa was separated fromthe IgG-binding moiety by a second passage through theIgG affinity column. Acute toxicity assay in micedemonstrated that the rBmK CTa was active (Fu et al.,2005).

Addional work has been performed such as theexpression of peptides responsible for insecticidal activityof scorpion toxin (ButaIT) and snowdrop lectin (GNA)containing fusion proteins toward pest species of differentorders (Fitches et al., 2010). All these designs rely ondifferent fusion proteins bound to the peptide of interest,working as a carrier of the spider peptide. One of thefunction of the carrier protein is to protect the spidercationic peptides against protease degradation (Piers et al.,1993). Additionally, the fusion system provides otheradvantages such as: 1) export out of the cytoplasmicenvironment; 2) affinity for a specific ligand, which enableseasy purification; and 3) the carrier protein could help inthe in vitro folding of the cysteine-rich spider polypeptides.Among the fusion proteins are glutathione S-transferase(GST) (Maggio and King, 2002; Tedford et al., 2004), thepresence of histidine tags, which permits an easy purifi-cation of the hybrid protein; thioredoxin fusion protein(Trx) (Ostrow et al., 2003) which helps on disulfide bridgeformation and facilitates folding efficiency. Otherconstructions have used green fluorescent protein (GFP)(Sanchez-Lopez et al., 2007). In summary, the use ofarachnid peptides fused to carrier proteins has becomea good strategy to reduce toxicity, increase yield andimprove biological activity by facilitating the correctdisulfide pairing and adequate folding.

8. Induction, purification, folding and biologicalactivity of scorpion and spider peptides

The induction of the expression of the protein of interestis certainly related to the characteristics of the chosenvector, but alternatively it is convenient to consider the useof a strong inducible promoter in order to easily regulatethe expression of the desired product. The moleculardesign details of a expression vector can be crucial to theprotein yield (Bass and Yansura, 2000). Upstream of themature sequences, it is usually suggested to use a histidinetag and a Factor Xa or Enterokinase recognition site, whichwill be important for protein purification using affinitychromatography and for releasing the desired peptide fromthe hybrid protein. Proper peptide folding is then requiredfor biological activity (Estrada et al., 2007; Hernández-Salgado et al., 2009).

Additional to the problems of expressing short peptides,as described above, another main difficulty is to obtain thepeptides correctly folded and capable of replicating iden-tical biological activity as the native ones, which weredirectly purified from the venoms of these arachnids. Veryfew examples have shown the correct expression andfolding of such peptides. Our group was able to properlyexpress one of the scorpion toxins as described before(Jímenez-Vargas et al., 2011). Another successful exampleof the expression of a scorpion peptide with correct foldingis the neurotoxin LqqV from Leiurus quinquestriatus quin-questriatus expressed as a poly-histidine-tagged fusionprotein in an E. coli strainwhich is a mutant for thioredoxin[AD494(DE3)pLysS], thus permitting disulfide bondformation. The fusion protein was cleaved by thrombin,resulting in a yield of about 1.5 mg/L. The structure of therecombinat LqqV and its funtional activity was confirmedby NMR, CD spectroscopy and electrophysiology (Banerjeeet al., 2006)

One of the main limitations in the expression of spidertoxins is the amount of active product. Although somereports have claimed a large production of spider toxins(Park et al., 2008), they have failed to prove the correctfolding and activity of these arachnid peptides. The incor-rect folding of expressed proteins is more the rule than theexception; so, authors should pay attention to the foldingprocess because incorrectly folded proteins result in low orno biological activity detected.

9. Conclusions and perspectives

From the venom of arachnids (scorpion and spiders)several hundred peptides have been isolated and charac-terized, most of which are relatively short peptides thatinterfere with cellular communication, either by blockingthe ion channels or modifying their gating mechanism,which cause abnormal cell depolarization and impairproper function. Polyamines and peptides with antimicro-bial, anti-parasitic, vasoconstrictive and analgesic activitieshave been described. A few enzymes have been alsocompletely characterized, although the venom of manyspecies of scorpions and the great majority of spider venomcomponents has not been studied. The newmethodologiesthat allow to obtain information by mass spectrometry and

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by cloning genes extracted from the venomous glands ofthese arachnids is contributing in a significant manner toadvance the knowledge in this field. Preparation of cDNAlibraries and transcriptome analysis has improved theamount of information lately obtained. Thousands of novelsequences have been obtained. From these cloning andsequencing systems it is evident that many more distincttypes of components are present in the venomous glands ofscorpion and spiders, apart from those that we alreadyknow. The newly described components need to be iden-tified directly from the venoms or need to be heterolo-gously expressed for verification of their function. This isnot a trivial task. It is necessary to improve the strategy forproper folding and to increase the final yields of recombi-nant products. Additional bioassays need to be developedfor finding new unknown functions of these components.The entire situation could be improved if at least one of thegenomes of any scorpions or spiders would be sequenced,assembled and completely annotated. The workingperspectives in this field of research are immense andcertainly in a short future we should overcome some ofthese difficulties.

Acknowledgments

This work was partially financed by grants from Direc-ción General de Asuntos del Personal Académico (DGAPA-UNAM) number IN204110 to LDP and IN220809 to GC.Additional support came from: CNPq/CONACyT (490068/2009-0) and CNPq (303003/2009-0 to EFS), and from theMexican company Instituto Bioclón S.A. de C.V. to LDP andBB.

Ethical statement

I declare that the work performed during the writing ofthe review entitled: “Scorpion and spider venom peptides:Gene cloning and peptide expression”, by Quintero-Her-nández, V., Ortiz, E., Rendon, M.R., Schwartz, E.F., Becerril,B., Corzo, G. and myself, now submitted for publication intothe journal TOXICON, if judged acceptable by the EditorialBoard, was performed within the strict guides of ethicaland respect. All authors read the manuscript and contrib-uted in their specialties to complete the work.

Conflict of interest statement

None declared.

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