Protein Expression and Purification 102 (2014) 38–44

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Protein Expression and Purification

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B-cell epitope of beta toxin of Clostridium perfringens geneticallyconjugated to a carrier protein: Expression, purification andcharacterization of the chimeric protein

http://dx.doi.org/10.1016/j.pep.2014.06.0141046-5928/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding authors. Tel.: +91 11 26704085 (A. Dixit). Tel.: +91 11 26703652;fax: +91 11 26742125 (L.C. Garg).

E-mail addresses: adixit7@gmail.com (A. Dixit), lalit@nii.ac.in (L.C. Garg).1 Abbreviations used: btx, beta toxin; LTB, B-subunit of heat labile enterotoxin.

Bharti Bhatia a, Amit Kumar Solanki a, Himani Kaushik a, Aparna Dixit b,⇑, Lalit C. Garg a,⇑a Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, Indiab School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 28 May 2014and in revised form 24 June 2014Available online 1 July 2014

Keywords:EpitopeEpitope prediction algorithmsBeta toxinClostridium perfringensLTBFusion proteinVaccine

Beta toxin (btx) is the prime virulence factor for the pathogenesis of Clostridium perfringens type C strain,known to cause necrotic enteritis and enterotoxaemia in mammalian species. The existing vaccines tar-geting btx are formaldehyde inactivated culture filtrates of Clostridium. These filtrates raise antigenic loadin the host leading to nonspecific and poor responses. The present study aimed to overcome these draw-backs and generate a chimeric protein carrying in silico identified B-cell epitope of btx fused with a carrierprotein as a vaccine candidate. Using bioinformatic tools, three stretches of amino acids were predicted asputative B-cell epitopes. One of the epitopes spanning 140–156 amino acid residues was genetically con-jugated with B-subunit of heat labile enterotoxin (LTB) of Escherichia coli and expressed as a translationalfusion in Vibrio cholerae secretory expression system. High level expression of the recombinant fusionprotein rLTB-Btx140–156 was obtained and the protein was successfully purified. The recombinant proteinretained the native LTB property to pentamerize and bind to GM1 ganglioside receptor of LTB. The anti-genicity of both the epitope and the carrier protein was maintained in fusion protein as indicated byimmunoblotting against anti-LTB and anti-btx antibody. The rLTB-Btx140–156 fusion protein thereforecan be evaluated as a potential vaccine candidate against C. perfringens.

� 2014 Elsevier Inc. All rights reserved.

Introduction

Clostridium perfringens type B and C strains have been impli-cated in enteritis necroticans in humans, lamb dysentery and ent-erotoxaemia. The sporadic outbreaks of the disease occur amongnew borns because of low synthesis of digestive enzymes. The casefatality rate varies, but 100% mortality in litters from non-immu-nized mothers is not unusual, and herd mortality is usually morethan 50% causing significant impact on the productivity of agricul-tural industry worldwide in terms of livestock loss [1]. The patho-genesis of C. perfringens is attributed mainly to the exotoxinssecreted; in case of C. perfringens type C strain, it has been estab-lished that the major virulence factor is beta toxin (btx)1 [2,3].

The disease progression is quite rapid and post infection recov-ery is rare, with survivors become unthrifty. After the appearanceof clinical signs of the disease, even the use of antibiotics offers a

limited help in controlling the disease. Therefore vaccination isconsidered the best defence against the disease. Conventional vac-cines targeting btx are formaldehyde inactivated culture filtrates ofClostridium. These filtrates are contaminated with many undesir-able proteins which raise antigenic load in the host leading touncontrolled inflammatory response and also have the risk ofreversion of inactivated toxin. Several attempts have been reportedto produce recombinant btx with various tags to aid purification[4,5]. Tagged proteins are not approved to use as vaccine [6].Therefore production of tag less recombinant proteins is essentialfor vaccine development as well as for therapeutic use.

A potential vaccine candidate is expected to elicit effectivehumoral immune response specifically against the pathogen. Inan antigen, the immunodominant surface epitopes are responsibleto generate B-cell mediated immune response, therefore are con-sidered to be an important constituent of a vaccine. A subunit vac-cine against a pathogen is based on such immunodominantepitopes together with a suitable adjuvant or a carrier protein.Genetic fusion of small peptides/epitopes to protein carriers hasbeen demonstrated to improve intrinsic immunogenicity of theepitopes and enabled their conformational expression [7–9]. Over

B. Bhatia et al. / Protein Expression and Purification 102 (2014) 38–44 39

the years, subunit vaccine development has become an integralpart of vaccine design in which immunogenic region of protein isused instead of complete pathogen or antigen which in turn makesvaccine safer.

In silico identification of epitopes is preferred over experimentalidentification as it is more economical in terms of time and effort.Bioinformatics tools enable researchers to move rapidly fromgenomic sequence towards vaccine design. These tools analyzevarious physicochemical properties of amino acids such as hydro-philicity, flexibility and surface accessibility to predict B- cell epi-topes. Further, to generate epitope specific high magnitude ofimmune response, the epitope is coupled with a suitable carrierprotein.

The present study demonstrates in silico identification of anti-genic domain of btx, its genetic conjugation with carrier proteinLTB and expression in Vibrio cholerae secretory expression system.The LTB of Escherichia coli used in the present study as a carrierprotein is structurally, functionally and immunologically similarto the CTB of V. cholerae and both are expressed with equal effi-ciency in V. cholerae [10,11]. Owing to the similarity of LTB withCTB, V. cholerae expression system can be used for the expressionof peptide bearing LTB hybrid proteins that have high immunoge-nicity due to the presence of LTB. Unlike Bacillus subtilis secretoryexpression system, V. cholerae allows the use of broad rangeE. coli promoters to express heterologous proteins [12,13]. Further,high level of LTB/CTB expression (�70% of the total secretory pro-teins) and efficient secretion of the same has been reported in V.cholerae [12,14,15], when compared to other secretory hosts suchas Saccharomyces cerevisiae in which the recombinant protein con-stituted only �0.5–1.3% of total cell free extract supernatant [16].Thus, the V. cholerae expression system offers a simple approachfor high level secretory expression and production of soluble LTBand btx epitope chimera without any purification tag.

Materials and methods

Materials, strains and cells

All chemicals were of analytical grade and purchased fromSigma–Aldrich, USA, unless stated otherwise. Restriction endonu-cleases and T4 DNA ligase were purchased from New EnglandBiolabs or Promega, USA. Gel extraction, DNA purification kitsand Ni–NTA beads were obtained from Qiagen, Germany.

In silico prediction of B-cell epitopes of btx

Online available bioinformatic tools Kyte Doolittle HydropathyPlot [17], Parker Hydrophilicity prediction [18], Bepipred epitopeprediction [19] and Emini Surface Accessibility Prediction [20]were used to predict linear B-cell epitopes of btx (NCBI AccessionNo. Q46308). Linear stretch of amino acid residues predicted bymost of the programs as epitope of btx were screened.

Generation of LTB-Epitope fusion construct

To generate recombinant fusion, complementary oligonucleo-tides corresponding to the amino acid sequences of the chosen epi-tope were synthesized along with a spacer of nucleotide sequencecoding for 5-glycine residues at the 50 end and termination codonat the 30 end. PstI and HindIII sites were included at the spacerand termination ends, respectively. A unique XhoI restriction sitewas also added just before the HindIII site for restriction analysisof the recombinants. The oligonucleotides were annealed andcloned into the SacI-PstI digested pQELTB plasmid available inour lab, carrying the LTB gene in plasmid pQE-32 vector [7]. The

ligated product was transformed into E. coli DH5a cells and theputative recombinants were screened by restriction digestion andconfirmed by automated DNA sequencing (DNA Sequencing Facil-ity, University of Delhi South Campus, New Delhi). The LTB-btx epi-tope insert was subsequently cloned into SacI and HindIII digestedpMMB secretory expression vector (derived from pMMB66EH vec-tor, GenBank Accession No. X15234). The resultant plasmid wasdesignated as pMMBLTB-Epi140–156.

Conjugal transfer of the pMMBLTB-Epi140–156 construct to V. choleraecells for secretory expression

E. coli DH5a cells harbouring the secretory expression plasmidpMMBLTB-Epi140–156 were conjugated with V. cholerae JBK70 cellsusing pRK2013 helper cells as described earlier [21]. Trans conju-gants were selected by double antibiotic selection in the presenceof both ampicillin (50 lg/ml) and polymyxin B (50 units/ml).

Expression and purification of fusion protein

V. cholerae cells harboring the recombinant plasmid pMMBLTB-Epi140–156 were induced with 1 mM IPTG (isopropyl-beta-D-thio-galactopyranoside) for 6 h and secretory expression of the recom-binant fusion protein was analyzed by SDS–PAGE (15%) of theculture supernatant. The recombinant fusion protein was purifiedfrom the culture supernatant as described by Mekalonos et al.[22] with minor modification. Briefly, induced culture cells wereharvested at 6000�g for 15 min at 4 �C. The supernatant wascollected and nonspecific proteins were salted out with 30%ammonium sulphate. The precipitated protein was removed bycentrifugation and the supernatant fraction was concentrated by70% ammonium sulphate precipitation. The pellet obtained after70% saturation was solubilized in 10 mM sodium phosphate bufferand loaded on to cellulose phosphate column. The protein waseluted by 300 mM sodium phosphate buffer. The fractions werecollected and analyzed on SDS–PAGE for the presence of purifiedprotein.

Western blot analysis

The protein resolved on SDS–PAGE was electrotransferred ontoa nitrocellulose membrane and was immunoblotted with primaryantibody and HRPO conjugated corresponding secondary antibodyessentially as described earlier [7,23]. Immunoreactive proteinbands were developed with di-amino-benzidine (0.5 mg/ml) andH2O2 (0.1%) in phosphate-buffered saline (PBS).

GM1 receptor binding assay

In vitro GM1 receptor binding activity of the fusion protein wasdetermined using sandwich ELISA as described earlier [23]. Immu-noplates were coated with 2 lg GM1 ganglioside receptor andincubated at 4 �C for 12–16 h. Nonspecific sites were blocked with3% milk in 50 mM PBS. Plates were then incubated with 5 ng/ll ofpurified rLTB-Btx140–156 fusion protein. 1% BSA and LTB proteinwere used as negative and positive controls, respectively. Logdilutions of rabbit anti-LTB antibodies and 1:5000 dilutions ofanti-rabbit IgG-HRP antibodies were then used to detect the boundprotein. The colour was developed by the addition of o-phenylene-diamine dihydrochloride as substrate. The absorbance was read at540 nm.

40 B. Bhatia et al. / Protein Expression and Purification 102 (2014) 38–44

Results and discussion

In silico prediction of B-cell epitopes of btx

C. perfringens btx is considered to be the potential immunother-apeutic molecule as it is the prime pathogenic factor of fatal necro-tic enteritis caused by C. perfringens type B and C strains indomestic animals. For the development of epitope based btx vac-cine, identification of immunodominant epitopes of btx is essen-tial. Prediction of linear B-cell epitopes of btx was done by usingmultiple bioinformatic algorithms such as Kyte Doolittle Hydro-phobicity plot, Parker Hydrophilicity prediction, Emini SurfaceAccessibility Prediction and Bepipred epitope prediction. All thesealgorithms access different physicochemical properties of aminoacid residues such as hydrophilicity, secondary structure and sur-face accessibility which are characteristic of a typical B-cell epi-tope. Therefore, prediction of an epitope using multiplealgorithms indicates that the predicted amino acid stretch wouldpossess more than one characteristics of B-cell epitope and in turnwill enhance the accuracy of prediction. Three stretches of aminoacids spanning from 32–45, 140–156 to 260–275 were predictedas putative epitopes by all the algorithms used (Fig. 1). In case ofKyte Doolittle Hydropathy Plot, low scores correspond to highhydrophilicity, indicating higher antigenic property of peptide.The sum of scores obtained from all the algorithms combinedwas highest for the region 140–156 as compared to the othertwo putative epitopes, and therefore this stretch of btx wasselected for generating fusion construct with LTB.

Generation of LTB-Epitope fusion construct pMMBLTB-Epi140–156

LTB, a homo-pentamer of 11.6 kDa polypeptide is a nontoxicpartner of E. coli heat labile enterotoxin. It has been reported thatLTB in its pentameric form binds to GM1 receptor of mammaliancells and enhance immunogenicity of the conjugated molecule[24–26]. Because of this exceptional adjuvant property, LTB is

Fig. 1. In silico analysis of linear B-cell epitopes of beta toxin. Different algorithms werParker Hydrophilicity prediction. (C) Emini Surface Accessibility Prediction. (D) BepiPred

considered to be a potent immunogen and therefore, was used asa fusion partner of in silico identified B-cell epitope of btx in thepresent study. C-terminus is preferred over N-terminus for makingtranslation fusion as this region of LTB has been reported to play animportant role in maintaining structural stability and GM1 receptorbinding property [27]. Moreover, N-terminal fusions have beenshown to have low secretory expression, poor pentamer formationand less efficient in receptor binding [28,29]. Nucleotide sequenceof btx epitope 140–156 with pentaglycine linker along withrestriction sites (Fig. 2A) was cloned at the C-terminus in transla-tion fusion with LTB in a secretory expression vector and the clon-ing strategy is shown in Fig. 2B. Glycine spacer allows independentmovement of the fusion partners to regain their original conforma-tion. The final construct was designated as pMMBLTB-Epi140–156.Linearization of pMMBLTB-Epi140–156 plasmid DNA by epitopespecific Xho I digestion (Fig. 3A, lane 2) and increase in ampliconsize with fusion gene specific primers as compared to LTB specificprimers (Fig. 3B, lane 3) indicated successful cloning. Automatedsequencing of the recombinant plasmid confirmed the successfulconstruction of the fusion gene construct.

Conjugal transfer of the pMMBLTB-Epi140–156 construct to V. choleraecells for secretory expression

Many heterologous proteins conjugated with LTB as fusion part-ner have been overexpressed and large-scale production has beenachieved using E. coli [13,30–32]. However, E. coli lacks the systemof extracellular transport due to which the transport of the pro-teins mainly occurs in periplasmic space or in case when proteinis over expressed, it often gets partially folded or misfolded andaccumulates as inclusion bodies in cytoplasm. V. cholerae, on theother hand, possesses main terminal branch of type II secretorypathway and has been shown to secrete some of its natural toxinsin extracellular milieu [33–35]. Vibrio secretory expression systemoffers the advantage of obtaining a refolded protein with high levelof expression in extracellular medium, eliminating the need to lyse

e used to predict surface exposed regions of beta toxin. (A) Kyte Doolittle plot. (B)linear epitope prediction.

Fig. 2. Cloning strategy for generation of Ltb-Btx140–156 fusion construct. (A) Nucleotide sequence of oligonucleotides corresponding to the beta toxin epitope withpentaglycine linker and restriction sites. (B) Pairs of oligonucleotides mentioned above were annealed and inserted in Pst I-Hind III digested pQE-LTB vector generating theconstruct pQELTB-Epi140–156. From this plasmid LTB-Linker-BtxEpi140–156 fragment was released and inserted into Sac I and Hind III digested pMMB plasmid. The finalconstruct pMMBLTB-Epi140–156 carried the translation fusion of btx epitope at C-terminal of LTB with 5-glycine residues in between under the control of Tac promoter. ‘T5’and ‘Tac’ indicate respective promoters. ‘SS’ indicates signal sequence of LTB.

Fig. 3. PCR and restriction digestion analysis of putative recombinants. (A) Lane Mdenotes DNA Marker; lanes 1 and 2 denote undigested and epitope specific Xho Idigested putative recombinant plasmid DNA, respectively. (B) Lanes 3 and 4 denotePCR amplified product obtained from the recombinant plasmid DNA using fusionspecific and LTB gene specific primers, respectively.

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the cells to obtain the protein of interest. The number of proteinssecreted in extracellular medium is very less which further easespurification.

For secretory expression of the recombinant fusion protein(rLTB-Btx140–156), fusion gene construct in E. coli was conjugallytransferred to V. cholerae cells. On SDS–PAGE analysis, a bandcorresponding to the expected size of fusion protein wasobserved in the culture supernatant of IPTG induced V. choleraecells harbouring the recombinant plasmid (Fig. 4A, lane 2). Nomajor band corresponding to the fusion protein was detected inuninduced culture (Fig. 4A lane 1). SDS–PAGE analysis ofunboiled sample of induced supernatant under non-reducingconditions showed a band of �45 kDa corresponding to LTB-Epi-tope fusion pentamer (Fig 4B, lane 1). However, 45 kDa band cor-responding to LTB-fusion pentamer got converted into �12 kDaband corresponding to LTB-fusion monomer when the samplewas analyzed under reducing conditions after boiling for 10 min(Fig. 4B, lane 2) suggesting that LTB has retained its ability topentamerize as fusion protein. Localization analysis of the fusionprotein confirmed that the induced protein is secreted to extra-cellular medium (Fig. 4C). These observations indicate that thefusion protein was successfully expressed and secreted to extra-cellular medium by Vibrio cells and LTB present in the fusion pro-tein retained the ability to pentamerize.

In order to purify the secretory fusion protein, ammonium sul-phate precipitation of the induced supernatant was carried outprior to ion exchange chromatography. SDS–PAGE analysis of puri-fied fractions showed an intense band suggesting efficient purifica-tion of fusion protein (Fig. 4D, lane 1). The yield of the recombinantprotein was approximately 30 mg/L of culture (Table 1).

Fig. 4. Expression, purification, localization and characterization of rLTB-Btx140–156 fusion protein. (A) Expression analysis of rLTB-Btx140–156 in V. cholerae. Lane M denotesprotein marker. Lanes 1 and 2 indicate uninduced and induced Vibrio cell culture supernatant, respectively. Arrow indicates monomeric form of fusion protein. (B) Analysis ofpentamer formation. Lanes 1 and 2 indicate unboiled and boiled samples, prepared in non-reducing and reducing conditions, respectively. The arrow head and arrow point tothe pentameric and monomeric forms of the fusion protein in unboiled and boiled samples, respectively. (C) Localization of fusion protein expression in V. cholerae. Lanes 1and 2 are the cell lysate and extracellular fractions of induced Vibrio cells culture. (D) SDS–PAGE analysis of the purified fusion protein. The purified fusion protein under nondenaturing conditions is shown in lane 1.

Table 1Purification of rLTB-Btx140–156.

S. No. Fraction Total protein (mg) Total activity a (U � 103) Specific activityb Fold purification % Yield

(1) Culture supernatant 73 102 1397 1.0 100(2) (NH4)2SO4 (70%) precipitate 33 �63 1909 1.37 �62(3) Purified protein (Cellulose phosphate column eluate) 29 �57 1965 1.40 �56

Induced culture supernatant was processed for purification.a In the absence of biological activity for the B cell epitope of beta toxin of C. perfringens in the fusion protein, the GM1 receptor binding activity of the LTB component in the

fusion protein was used to monitor the purification yields at different steps of purification. One unit (U) of the rLTB-Btx140–156 is defined as the amount of protein required togive rise absorbance equivalent to 500 ng of purified LTB in a GM1 receptor binding activity assay under experimental conditions.

b Specific activity is the units per milligram protein.

42 B. Bhatia et al. / Protein Expression and Purification 102 (2014) 38–44

Antigenic analysis of fusion protein

To determine the antigenicity of the recombinant fusion proteinrLTB-Btx140–156, Western blot analysis of the fusion protein wascarried out using antisera raised against LTB and btx. The fusionprotein was recognized by both anti-LTB and anti-btx antibodies,respectively (Fig. 5A and B) suggesting that the antigenicity ofLTB as well as the B-cell epitope of the btx was preserved in the

Fig. 5. Antigenic analysis of fusion protein by Western blotting. (A) Lanes 1 and 2correspond to the rLTB and rLTB-Btx140–156 fusion protein transferred ontonitrocellulose membrane, respectively and immunoblotted with anti-rLTB anti-body. (B) Lanes 1 and 2 correspond to the rLTB-Btx140–156 fusion protein and rbtxtransferred onto nitrocellulose membrane, respectively and immunoblotted withanti-btx antibody. M in both panels indicates protein molecular weight marker.

recombinant fusion protein. Slower migration of the recombinantfusion protein in comparison to LTB is an indication of increasein molecular weight of fusion protein due to the addition of thebtx epitope at the C-terminus of LTB. Western blot analysis alsoconfirmed the authenticity of LTB and B-cell epitope as fusion part-ners and established that the antibodies against region of 140–156amino acid residues chosen as an epitope of btx in the study waspresent in the sera raised against btx indicating it to be one ofthe dominant epitopes of btx.

GM1 receptor binding of fusion protein

In order to evaluate the receptor binding activity of the purifiedrecombinant fusion protein, ELISA based GM1 ganglioside receptorbinding assay was carried out. GM1 receptor binding assayrevealed that receptor binding of rLTB-Btx140–156 fusion protein issimilar to that of the native LTB, however, no binding was observedwith BSA which served as negative control (Fig. 6). These resultssuggest that the translational fusion of btx epitope with LTB didnot affect the receptor binding activity of LTB. It has been demon-strated that LTB binds to GM1 ganglioside receptor after attainingits native conformation of pentameric structure. The efficient bind-ing of the recombinant fusion protein clearly indicated that thefusion protein expressed in secretory V. cholerae expression systemwas able to fold into its native conformation as pentamer. It hasbeen established that GM1 binding activity is essential and suffi-cient for immunogenicity of LTB [36], therefore the fusion proteinis expected to be highly immunogenic.

In brief, rLTB-Btx140–156 fusion protein is expressed in nativeconformation in extracellular medium by Vibrio secretory system

Fig. 6. GM1 ganglioside receptor binding assay of rLTB-Btx140–156 fusion protein.Binding of the rLTB-Btx140–156 fusion protein to GM1 ganglioside was checked bysandwich ELISA using anti-LTB antibodies. LTB and BSA were used as positive andnegative controls, respectively.

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and therefore, easy to purify in large amount without tag. More-over, it is highly immunogenic and being homogenic in purifiedform, it is more specific than the conventional culture filtrates ofC. perfringens. Hence, rLTB-Btx140–156 fusion protein is a potentialvaccine candidate against btx of C. perfringens.

Conclusion

In conclusion, a fusion construct of LTB and B-cell epitope of C.perfringens btx was made and expressed in extracellular milieuusing V. cholerae secretory expression system. LTB in the purifiedretained its ability to pentamerize as well as its ability for receptorbinding. The fusion protein maintained the antigenicity of the B-cell epitope and carrier protein, therefore can be used as potentialmucosal vaccine against btx. Additionally the identified epitopecould also serve as a target for generating monoclonal antibodiesagainst the toxin for diagnostic purposes.

Acknowledgments

This work was supported by research Grant from Department ofBiotechnology, New Delhi. The Council of Scientific and IndustrialResearch, New Delhi and Department of Biotechnology, New Delhiare acknowledged for research fellowship to BB and AKS, respec-tively. The authors thank Mr. K.P. Pandey for his technicalassistance.

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