BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Expression, purification, and immunogenic characterizationof Epstein–Barr virus recombinant EBNA1 proteinin Pichia pastoris

Man Wang & Shuai Jiang & Xiaoying Liu & Yefu Wang

Received: 6 March 2013 /Revised: 25 April 2013 /Accepted: 29 April 2013 /Published online: 18 May 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Epstein–Barr virus (EBV) is a ubiquitous humanherpesvirus associated with the development of both lymphoidand epithelial tumors. EBNA1 is the only viral proteinexpressed in all EBV-associated malignancies and plays impor-tant roles in EBV latency. Thus, EBNA1 is thought to be apromising antigen for immunotherapy of all EBV-associatedmalignancies. This study was undertaken to produce recombi-nant EBNA1 protein in Pichia pastoris and evaluate its immu-nogenicity. The truncated EBNA1 (E1ΔGA, codons 390–641)was expressed as a secretory protein with an N-terminal histi-dine tag in the methylotrophic yeast P. pastoris and purified byNi-NTA affinity chromatography. The purified proteins werethen used as antigens to immunize BALB/cmice for productionof polyclonal antibodies. Western blot analysis showed that thepolyclonal antibodies specifically recognized the EBNA1 pro-tein in B95-8 cell lysates. The recombinant E1ΔGA alsoinduced strong lymphoproliferative and Th1 cytokine re-sponses in mice. Furthermore, mice immunized with E1ΔGAdeveloped CD4+ and CD8+ T cell responses. These findingsshowed that the yeast-expressed E1ΔGA retained good immu-nogenicity and might be a promising vaccine candidate againstEBV-associated malignancies.

Keywords Epstein–Barr virus (EBV) . EBNA1 . Pichiapastoris . Immunogenicity . Polyclonal antibodies . Vaccine

Introduction

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirusthat latently infects B cells and establishes chronic infection inmore than 90 % of the world population (Conacher et al. 2005;

Gurer et al. 2008; Herrmann and Niedobitek 2003). EBV is thecausative agent of infectious mononucleosis and is closely asso-ciated with a number of malignancies including Hodgkin's dis-ease, Burkitt's lymphoma, post-transplant lymphoproliferativedisorder, and nasopharyngeal carcinoma (NPC) (Fu et al. 2004;Thorley-Lawson 2001). EBV nuclear antigen 1 (EBNA1) isessential for replication of EBV episome and maintenance oflatency (Kitamura et al. 2006). Aside from its role in viralpersistence, EBNA1 also alters cellular processes in ways thatcontribute to host cell survival and proliferation (Cao et al. 2012).First, EBNA1 is expressed in all forms of latency in proliferatingcells and is the only EBV protein consistently expressed in allEBV-associated malignancies (Babcock et al. 2000; Frappier2012; Sivachandran et al. 2008). In addition, specific knockdownof EBNA1 expression by RNA interference in Raji Burkitt'slymphoma (Hong et al. 2006) and NPC cells (Yin andFlemington 2006) has been found to decrease cell proliferation.

EBNA1 comprises 641 amino acids and is amultifunctional DNA-binding protein (Mayer et al. 2012).The N-terminus of EBNA1 contains two chromatin associ-ation domains, which are separated by a large glycine–alanine repeat (GAr) region (Lindner and Sugden 2007;Mackey and Sugden 1999). The GAr domain inhibitsproteosomal degradation of EBNA1 and thereby preventsantigen processing and presentation of EBNA1 epitopes onthe cell surface (Fogg et al. 2005; Levitskaya et al. 1997).The C-terminus of EBNA1 contains DNA-binding anddimerization domains. As a DNA-binding protein, EBNA1can also transactivate the expression of other EBV latentgenes (Gahn and Sugden 1995). Since EBNA1 expression ismaintained even in the absence of all other EBV proteins,EBNA1 can serve as an important target antigen for immu-notherapy (Voo et al. 2005). Increasing numbers of obser-vations suggest that T cell responses to EBNA1 areimportant in eliminating virally infected B cells and control-ling EBV infection (Lee et al. 2004; Ruiss et al. 2011; Tsanget al. 2006).

M. Wang : S. Jiang :X. Liu :Y. Wang (*)State Key Laboratory of Virology, College of Life Sciences,Wuhan University, Wuhan 430072, People’s Republic of Chinae-mail: wangyefu@whu.edu.cn

Appl Microbiol Biotechnol (2013) 97:6251–6262DOI 10.1007/s00253-013-4967-x

To develop an effective vaccine for EBV-associated tu-mors, it is important to obtain large quantities of purifiedEBNA1 in a suitable heterologous expression system.EBNA1 has been expressed in recombinant baculovirus-infected Sf9 insect cells (Meij et al. 2000; Steigerwald-Mullen et al. 2000) and E. coli (Bouallag et al. 2009;Duellman and Burgess 2006). However, EBNA1 containsa large number of rare codons which can significantly re-duce expression levels (Bouallag et al. 2009; Mayer et al.2012). Codon optimization is a promising technique forincreasing protein expression level (Teng et al. 2007). Themethylotrophic yeast Pichia pastoris has become an impor-tant host organism for the high-level production of recom-binant proteins (Abad et al. 2011; Cereghino and Cregg2000; Cregg et al. 2000). In this study, the C-terminalfragment of EBNA1 (E1ΔGA) was optimized andexpressed according to its preferred codon usage in P.pastoris. More importantly, the recombinant E1ΔGA elicitedstrong humoral and cellular immune responses, suggestingthat the yeast-expressed E1ΔGA retained good immunoge-nicity. This study showed that P. pastoris production system issuitable for high-level expression of E1ΔGA through codonoptimization and a prerequisite for large-scale industrialproduction of a subunit vaccine candidate.

Materials and methods

Strains, plasmids, enzymes, and cell line

The P. pastoris strain GS115 (His4 deficient) and the expres-sion vector pPICZαA purchased from Invitrogen (USA) wereused as the expression hosts. The Escherichia coli strainDH5α, the vector pUC57, and enzymes were purchased fromTakara Bio Inc. (Dalian, China). E. coli cells with plasmidswere cultured at 37 °C in Luria–Bertani medium (yeast extract,5 g/L; tryptone, 10 g/L; NaCl, 10 g/L; agar, 15 g/L) containing25 μg/mL Zeocin (Invitrogen, USA). The EBV producingB95-8 cells (ATCC CRL 1612) were cultured in RPMI 1640medium supplemented with 10 % fetal bovine serum in 5 %CO2 incubator at 37 °C.

Codon optimization and construction of pPICZαA–E1ΔGAexpression vectors

The codon-optimized gene was designed based on the pro-tein sequence of EBNA1 (GenBank accession numberAFJ06864) (Kwok et al. 2012) according to the codon biasof P. pastoris (http://www.kazusa.or.jp/codon) (Bai et al.2011). Codon optimization was performed by using JCatprogram (Grote et al. 2005). The truncated EBNA1 (codons390 to 641, henceforth referred to as E1ΔGA) with an N-terminal His6-tag was synthesized by Sangon Biological

Co., Ltd. (Shanghai, China). One stop codon was added atthe C terminus to form a termination signal. The syntheticgene was ligated into pUC57 plasmid, resulting in pUC57–E1ΔGA. The E1ΔGA gene fragment was released from thepUC57–E1ΔGA vector by digestion of EcoRI/XbaI andsubcloned into the P. pastoris expression vector pPICZαAalong with the open reading frame of the α-factor signal,which is under the control of the AOX1 promoter (Fig. 1a).The recombinant pPICZαA vector was transformed into theE. coli DH5α competent cells. The positive clonescontaining pPICZαA–E1ΔGA were screened with Zeocinas the selective agent and identified by restriction analysisand sequencing.

Expression and purification of recombinant protein

The recombinant vector pPICZαA–E1ΔGA was linearizedwith SacI and electroporated into competent P. pastorisGS115 cells. pPICZαA–E1ΔGA-positive transformantswere screened on YPD plates containing 0.1 mg/mL Zeocin,and the presence of E1ΔGA insert in these clones wasfurther confirmed by genomic PCR using the 5′ AOX1primer 5′-GACTGGTTCCAATTGACAAGC-3′ and the 3′AOX1 primer 5′-GCAAATGGCATTCTGACATCC-3′. ThePCR-positive transformants exhibiting resistance to0.1 mg/mL Zeocin were inoculated into 25 mL bufferedcomplex glycerol media (BMGY) with vigorous shaking(300 rpm) at 30 °C until the culture reached OD600=6.The cells were harvested by centrifugation at 3,000×g for5 min and resuspended to OD600 of 1 in 1 L bufferedminimal methanol media (BMMY) in 5-L flasks to induceexpression. The flasks were incubated at 30 °C, and meth-anol was added to a final concentration of 0.5 % every 24 hto sustain the induction. A negative control containing theempty pPICZαA vector was carried out in parallel. After72 h of induction, the culture supernatant was collected andsubjected to 80 % ammonium sulfate precipitation. Theprecipitated protein was collected by centrifugation at10,000×g for 15 min, dissolved in distilled water, and dia-lyzed extensively against 50 mM Tris-buffered saline (TBS,pH 7.2) at 4 °C overnight by changing the dialysis buffer threetimes. The dialysate was loaded onto a Ni-NTA affinity col-umn (GE Healthcare), and the bound proteins were eluted by0.2 M imidazole in 50 mM TBS (pH 7.2) after extensivewashing with TBS. Protein concentration was determinedusing the bicinchoninic acid (BCA) Protein Assay Kit withbovine serum albumin (BSA) as a standard (Pierce, USA).Briefly, a stock solution of 2 mg/mL BSA was sufficientlydiluted to prepare a set of standards. For each protein sample,multiple dilutions were prepared in phosphate-buffered saline(PBS). A total of 25 μL of the standards and sample dilutionswas transferred into a 96-well microtiter plate containing200 μL of the mixture of BCA kit reagents. All sample

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dilutions were run in triplicate. The plate was incubated at37 °C for 30 min and measured at 562 nm.

Molecular weight determined by MS

The purified E1ΔGA was dissolved in a saturated solutionof α-cyano-4-hydroxycin-namic acid containing 50 %acetonitrile and 0.1 % TFA, then deposited on the target,and analyzed on a MALDI-TOF mass spectrometer(PerSeptive Bio-systems) (Woo et al. 2001).

Immunization of mice and serological test

Three groups, each including ten 4–6-week-old femaleBALB/c mice, were immunized subcutaneously on day0 with PBS (100 μl), 10 μg E1ΔGA, or 10 μg E1ΔGAwith Freund's complete adjuvant (50 %, v/v) (Sigma, USA).At 2-week intervals, the mice were boosted twice with PBS(100 μl), 10 μg E1ΔGA, or 10 μg E1ΔGA with Freund'sincomplete adjuvant (50 %, v/v) (Sigma, USA). Sera werecollected on days 0, 14, 28, and 42. Serum levels of IgG andIgM antibodies against E1ΔGA were measured by ELISA.Briefly, 96-well plates coated overnight with 50 ng purifiedE1ΔGA in 100 μl of 100 mM carbonate buffer (pH 9.6) perwell at 4 °C were washed with PBST (PBS containing 0.1 %Tween 20) and blocked with 2 % BSA in PBST. The plateswere subsequently incubated with diluted sera for 1.5 h atroom temperature. After three washes with PBST, HRP-conjugated goat anti-mouse IgG or IgM (1:5,000) wasadded into each well. The plates were developed withtetramethylbenzidine substrate (Sigma, USA), stopped with2 M H2SO4, and measured at 450 nm. All samples were runin triplicates.

Western blot analysis

Mice sera collected on day 42 were used for western blotanalysis. Proteins were separated on 12 % SDS-PAGE,transferred onto PVDF membranes, and probed with 1:500diluted mice sera. Mice antibodies were detected with thecorresponding HRP-conjugated secondary antibodies(Sigma, USA), and the signals were developed byECL (Pierce, USA).

Lymphoproliferation assay

The spleens were aseptically removed from mice (n=3–5 eachgroup) at 2 weeks after the last immunization. Lymphocyteswere separated by an EZ-SepTM mouse lymphocyte separationkit (Dakewe, China). The isolated splenocytes were seeded intoa 96-well plate at a density of 1×106 cells per well and culturedwith purified E1ΔGA (5 μg/mL), Con A (5 μg/mL, Sigma,USA), or medium alone (negative control) at 37 °C for 72 h.Proliferative activity was measured using the MTT assay asdescribed (Kumar et al. 2010). The stimulation index wascalculated as the ratio of the average OD570 value of wellscontaining antigen-stimulated cells to the average OD570 valueof wells containing only cells with medium. All assays wereperformed in triplicate.

Cytokine assays

Cytokines were measured by ELISA as described previous-ly (Islam et al. 2005; Liu et al. 2008). Splenocytes fromimmunized mice were cultured in the presence of differentstimuli as described for the lymphoproliferation assay. At72 h post-incubation, cell-free supernatants were harvested

Fig. 1 Construction of the pPICZαA–E1ΔGA expression vector. aSchematic diagram of pPICZαA–E1ΔGA construction and transfor-mation into P. pastoris GS115. The C-terminal fragment of EBNA1(codons 390–641), with a His6-tag at 5′ end, was inserted intopPICZαA for the expression of recombinant protein. b PCR screening

analysis of the transformants obtained on YPD plates (lanes 2-7)indicated which colonies had integrated the gene encoding E1ΔGAinto the P. pastoris genome. A colony transformed by empty pPICZαAvector (lane 1) was used as a negative control

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and assayed for IFN-γ, TNF-α, IL-4, and IL-10 productionby commercially available ELISA kits (BD Biosciences).

Flow cytometry analysis

Splenocytes and PBMCs were isolated from mice (n=3–5each group) at 3 days or 6 days after the last immunization.A total of 1×106 cells/sample were stained with FITC-conjugated anti-CD3 and PE-conjugated anti-CD4 or PE-conjugated anti-CD8 (BD Biosciences). All samples wereanalyzed by flow cytometry (BD Biosciences).

Statistical analysis

All statistical analyses were performed using GraphPadPrism 5 software (GraphPad, San Diego, CA, USA).P-values less than 0.05 were considered as statisticallysignificant.

Nucleotide sequence accession number

The nucleotide sequence of E1ΔGA gene has been depositedin the GenBank database under accession number KC758960.

Results

Construction and transformation of pPICZαA–E1ΔGA

The length of E1ΔGA gene was 759 bp, which encoded 252amino acids. To improve the expression efficiency of therecombinant protein, the DNA sequence encoding E1ΔGAwas designed and synthesized based on the codon bias of P.pastoris. A total of 161 codons (almost 63.9 %) weresubstituted by the P. pastoris preferred codons. The opti-mized E1ΔGA gene was then inserted into the plasmidpPICZαA for protein expression, designated as pPICZαA–E1ΔGA (Fig. 1a). DNA linearized by SacI was transformedinto P. pastoris GS115 cells by electroporation. Thetransformants were sequentially screened by PCR amplifi-cation. As shown in Fig. 1b, the gene encoding E1ΔGAwasamplified from five of the six colonies.

Expression of E1ΔGA in P. pastoris

The P. pastoris GS115 cells harboring pPICZαA–E1ΔGAwere induced with methanol for 72 h. A protein band ofapproximately 28 kDa was observed in the supernatant offour positive transformants (pPICZαA–E1ΔGA/GS115)(Fig. 2a), and the size was consistent with the E1ΔGA mass(27,675.530 Da) identified by MALDI-TOF MS (data notshown). The band could not be detected in the supernatantof pPICZαA/GS115 (Fig. 2, lane 1). The expression of

E1ΔGA protein was verified by western blot analysisusing rabbit anti-His polyclonal antibodies, and the ex-pression levels of E1ΔGA were similar for all fourcolonies (Fig. 2b).

Production of E1ΔGA in large-scale shake flasksand protein purification

The E1ΔGA-positive transformant was cultured in a shakeflask to measure E1ΔGA expression. Maximum expressionwas observed after 3 days of methanol induction at 30 °C.Five 5-L flasks each containing 1 L BMMY were inoculatedwith 25 mL BMGY containing overnight-incubatedpositive-transformant seeds at 30 °C and constantlyincubated for 3 days. The induced supernatant wascollected for SDS-PAGE and western blot analysis(Fig. 3b, c).

The supernatant containing the secreted E1ΔGA washarvested, and ammonium sulfate was added to precipitatethe proteins. The protein sample was loaded onto the Ni-NTA affinity column and eluted with 0.2 M imidazole afterextensive TBS washing (Fig. 3a). A total fraction of 10 mLeluate (67–77 mL) was collected. Representative purifica-tion fraction from the affinity column was run on 12 % SDS-PAGE. The gel showed a single band of approximately28 kDa in the elution fraction (Fig. 3b, lane 3). The elutedprotein showed 95 % or more purity by electrophoreticanalysis with 12 % SDS-PAGE as analyzed by the Bandscansoftware (BioMarin Pharmaceutical Inc, UK) (Fig. 3b, lane 3).Table 1 showed the yield of E1ΔGA at each purification step,and the final production yield was 210.53 mg/L of culturesupernatant. In addition, western blot analysis showed thatanti-His polyclonal antibodies were able to detect the purifiedE1ΔGA (Fig. 3c, lane 3).

Humoral immune response in mice against the recombinantE1ΔGA

Groups of BALB/c mice were immunized three times at 2-week intervals with PBS, purified E1ΔGA, or purifiedE1ΔGAwith Freund's adjuvant (FA). The sera were collect-ed from mice at the indicated time points. The sera collectedon day 42 were subjected to western blot analysis to evalu-ate anti-E1ΔGA antibodies. The E1ΔGA protein bandswere detected with the antisera from E1ΔGA- orE1ΔGA+FA-immunized mice at a dilution of 1:500, whileE1ΔGA could not be detected with antiserum from PBS-immunized mice (Fig. 4a). Additionally, pre-immune serumfrom each group was not able to detect the E1ΔGA protein.The anti-E1ΔGA polyclonal antibodies were able to recog-nize the EBNA1 protein in cell lysates of B95-8 (Fig. 4b).

As shown in Fig. 5a, significantly high levels of IgGantibodies were detected in the sera of E1ΔGA- and

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E1ΔGA+FA-immunized groups after the primary immuni-zation, and the levels of IgG in the two groups increasedwith successive immunizations. E1ΔGA immunization alsotriggered significantly higher IgM levels than the controlgroup after booster injections (p<0.05; Fig. 5b). All vacci-nated mice remained healthy, and none of them exhibitednoticeable side effects during the course of the study.

E1ΔGA-induced cellular immune response in mice

The splenocytes from immunized mice were prepared at2 weeks after the last immunization to assess the proliferativeimmune responses to E1ΔGA. The splenocytes of E1ΔGA-or E1ΔGA+FA-immunized mice showed a greater increasein cell proliferation than the control group (p<0.01; Fig. 5c).

Fig. 2 Analysis of E1ΔGA expression in P. pastoris. The expressionof E1ΔGA in P. pastoris transformants was analyzed by SDS-PAGE(a) and western blotting using rabbit anti-His polyclonal antibodies (b).M protein markers; lane 1 negative control, induced supernatant of

control transformant (pPICZαA/GS115); lanes 2-5 induced supernatantof positive transformants (pPICZαA–E1ΔGA/GS115). The position ofE1ΔGA is indicated. The signal intensity of each band was quantifiedusing Image J software

Fig. 3 Expression andpurification of E1ΔGA. Thecrude proteins in the inducedsupernatants were precipitated,dissolved, and loaded onto aNi-NTA column. After washingwith TBS, E1ΔGA was elutedwith TBS containing 0.2 Mimidazole (indicated by anarrow). The image wasgenerated by AKTA prime(Amersham Biosystems) (a).The protein samples wereanalyzed by SDS-PAGE (b) andwestern blot with anti-Hisantibody (c). M proteinmarkers; lane 1 negativecontrol, induced supernatant ofcontrol transformant(pPICZαA/GS115); lane 2induced supernatant of positivetransformant (pPICZαA–E1ΔGA/GS115); lane 3purified E1ΔGA protein. Theposition of E1ΔGA is indicated

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Profiles of cytokine released from E1ΔGA-stimulatedsplenocytes were examined to determine the primed path-way of immune response. The Th1 (IFN-γ and TNF-α) andTh2 (IL-4 and IL-10) cytokines in the supernatant ofsplenocyte cultures were measured by ELISA (Fig. 5d).Splenocytes of mice immunized with E1ΔGA orE1ΔGA+FA produced higher levels of IFN-γ and TNF-αthan the control mice (p<0.01). However, injections ofE1ΔGA or E1ΔGA+FA in mice did not cause obviouschanges in the levels of IL-4 and IL-10. These resultsconfirmed that the cellular immune response induced bythe recombinant E1ΔGA was inclined to a Th1 typeresponse in mice.

Proliferation of T cell populations in immunized mice

To verify whether E1ΔGA could induce T cell responses,we detected T cell populations in spleens and peripheralblood of immunized mice. FACS analysis of the spleen Tcell populations showed that there were no differences in theproportions of CD4+ and CD8+ T cells among the threegroups (Fig. 6a). In contrast, the proportions of these cellswere slightly reduced in blood of E1ΔGA- or E1ΔGA+FA-immunized mice compared with the control mice (Fig. 6b).The absolute numbers of splenocytes and PBMCs werefound to be significantly increased in E1ΔGA- andE1ΔGA+FA-immunized mice (Fig. 6c, d). Therefore,higher numbers of CD4+ and CD8+ T cells were observedin spleen and peripheral blood of E1ΔGA- or E1ΔGA+FA-immunized group than the control group (Fig. 6e, f). Ourfindings suggested that the recombinant E1ΔGA couldefficiently induce CD4+ and CD8+ T cell responses.

Discussion

EBV infection is associated with multiple human malignan-cies (Xu et al. 2008). Vaccines that either prevent EBVinfection or lower the viral load might reduce certain EBVmalignancies. One of the urgent goals of EBV research is todevelop an effective vaccine. For an EBV vaccine to beeffective against virus-associated cancers, it should be ableto induce a robust T cell-mediated immune response (Cohenet al. 2011). Lots of studies have shown that EBNA1 is animportant target for both CD4+ and CD8+ T cell responses(Munz et al. 2000; Tellam et al. 2004), and EBNA1-specificT cell responses can eliminate EBV-associated tumor cells(Demachi-Okamura et al. 2008; Paludan et al. 2002; Voo etal. 2002). Therefore, EBNA1 is thought to be a promisingtarget for vaccine development against EBV-associated ma-lignancies. Recently, an EBNA1–LMP2 chimeric proteinthat induced T cell responses has completed phase I trialsin NPC patients (Long et al. 2011).

This study aimed to achieve a high-level expression ofrecombinant E1ΔGA protein in P. pastoris for developinguseful and cheaper subunit vaccines and made importantprogresses. The methylotrophic P. pastoris has been suc-cessfully used as a host for the expression of heterologousgenes (Daly and Hearn 2005), and accordingly, the produc-tion of E1ΔGA in yeast rather than in bacteria orbaculovirus may be preferable. The expression system ofP. pastoris offers several advantageous features includinglow cost, ease of genetic manipulation, stable expression,and rapid growth rate (Lu et al. 2013). Foreign genes in P.pastoris can be induced to a high level of expression, andthe expressed proteins can be subjected to post-translationalmodifications, which is important in the recombinant ex-pression of eukaryotic proteins (Fong et al. 2008). P.pastoris has a number of strong promoters available to driveheterologous expression (Cereghino and Cregg 2000;Macauley-Patrick et al. 2005). Heterologous gene expres-sion in P. pastoris is often performed under the control ofthe tightly regulated methanol-induced alcohol oxidase 1promoter (pAOX1) (Abad et al. 2010; Fairlie et al. 2000),

Table 1 Purification of recombinant E1ΔGA

Fraction Total protein (g) Volume (L) Yield (%)

Culture supernatant 3.11 3.23 100

(NH4)2SO4 1.59 1.05 51.13

Ni-NTA column 0.68 0.05 21.87

Fig. 4 The immunogenicityand specificity of therecombinant E1ΔGA. PurifiedE1ΔGA (a) and cell lysates ofB95-8 cells (b) were resolvedby 12 % SDS-PAGE,transferred onto PVDF, andreacted with mice sera collectedon day 42. E1ΔGA+FArecombinant E1ΔGA proteinwith Freund's adjuvant

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and pAOX1 can be highly induced by methanol (Pan et al.2012; Resina et al. 2004). Meanwhile, P. pastoris has beenreported as an excellent secretor of heterologous proteinsinto the culture medium (Yegin and Fernandez-Lahore2013). The secretory expression of recombinant proteinscan avoid the intracellular accumulation of target protein(Damasceno et al. 2012). P. pastoris secrets only smallamounts of endogenous proteins, so the secreted recombi-nant protein constitutes the vast majority of the total proteinin the medium, which makes the purification process easier(Lin-Cereghino et al. 2013). In addition, P. pastoris systemhas demonstrated its usefulness as a large-scale fermentationtool for the production of recombinant foreign proteins(Cregg et al. 2000). Since the C-terminal domain of EBNA1contains the major immunogenic epitopes (Chen et al.1993), we used P. pastoris yeast to express a truncated formof EBNA1 protein (E1ΔGA), which consists of EBNA1residues 390–641, and we successfully expressed E1ΔGAin P. pastoris to a high level via codon optimization andmethanol induction, indicating that the optimization strate-gies were useful in E1ΔGA high-level expression. More-over, the secreted E1ΔGA was easily purified from culturesupernatants by using Ni-NTA affinity chromatography.Several expression systems have been successfully used to

produce EBNA1 such as E. coli (Mayer et al. 2012), insectcells (Meij et al. 2000), and yeast (Hu et al. 2007). About0.8 mg purified EBNA1 was obtained from 6×107 insectcells (1 L). In E. coli expression system, the yield of EBNA1protein was only 2 mg/L of culture medium. In the presentstudy, the yield of purified E1ΔGA was estimated to be210.53 mg/L, which was significantly higher than thatobtained in E. coli or insect cells. The wild type of EBNA1has previously been expressed in P. pastoris, and the con-centration of wtEBNA1 was estimated to be 33.5 mg/L inculture supernatant (Hu et al. 2007). Compared with theexpression of the wild-type gene, this optimized E1ΔGAgene was expressed in P. pastoris at 6.3-fold-higher level.

In the past decade, P. pastoris has been geneticallyengineered to produce hundreds of recombinant proteinsimportant to industrial, pharmaceutical, and basic researchpurposes (Bollok et al. 2009). A number of different genesachieved high-level or increased expression through codonoptimization, such as Bacillus licheniformis beta-1,3-1,4-glucanase (Huang et al. 2008; Teng et al. 2007), Aspergillussulphureus endo-beta-1,4-xylanase (Li et al. 2010),Thermotoga maritima β-fructosidase (Menendez et al.2013), Coprinus cinereus peroxidase (Kim et al. 2009),Gymnema sylvestre gurmarin (Sigoillot et al. 2012),

Fig. 5 Humoral and cellular immune responses in vaccinated mice.IgG (a) and IgM (b) antibodies induced by E1ΔGA immunizationwere detected by ELISA. The sera of a and b were diluted 320- and160-fold, respectively. c Splenocyte proliferation assay. Splenocytes(106 cells/well) from mice were incubated in vitro with purifiedE1ΔGA, Con A (positive control), or medium (negative control).

d Cytokine production by splenocytes from immunized mice.After 72 h of incubation, concentrations of IFN-γ, TNF-α, IL-4,and IL-10 in the supernatants of splenocyte cultures were deter-mined by ELISA. All data presented were mean±SD of threereplicate wells (*p<0.05; **p<0.01; ***p<0.001)

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Fig. 6 Proliferation of T cell subsets in E1ΔGA-immunized mice.Pooled splenocytes and PBMCs from mice were collected, stainedwith fluorescence-labeled anti-CD3, anti-CD4, or anti-CD8, and ana-lyzed by flow cytometry. Representative FACS plots for single-cellsuspensions in spleen (a) and peripheral blood (b) are shown. Absolutecell numbers of splenocytes (c) and PBMCs (d) in immunized mice

were calculated using a hemocytometer. Relative frequencies of T cellpopulations in spleen (e) and peripheral blood (f) were calculated as theratio of the average number of CD4+ or CD8+ T cells in E1ΔGA- orE1ΔGA+FA-immunized mice to the average number of that in thecontrol mice

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eukaryotic membrane proteins (Oberg et al. 2011), andwheat puroindoline-a (Issaly et al. 2001). Some humangenes including glucocerebrosidase (Sinclair and Choy2002), adenosine A2A receptor (Singh et al. 2008), activinA (Fredericks et al. 2010), and Zbtb7A (Wang et al. 2008)were highly expressed in P. pastoris by codon optimization.P. pastoris expression system has also been widely used inviral protein production (Li et al. 2012). For example, thecodon-optimized capsid protein (opti-Cap) of porcinecircovirus type 2 has been highly expressed in P. pastoris,and the recombinant opti-Cap showed good immunogenic-ity (Tu et al. 2012). Hepatitis B e antigen (HBeAg) has beensuccessfully expressed in P. pastoris, and the yield ofcodon-optimized HBeAg was higher than the wild type ofHBeAg. Also, the P. pastoris-derived HBeAg showed highspecificity and sensitivity for serological tests of hepatitis Bvirus (Li et al. 2008). The modified ORF6 gene of porcinereproductive and respiratory syndrome virus was expressedin P. pastoris with a higher level, but the native ORF6 genecould not be expressed. The recombinant product of themodified ORF6 gene can provide a tool for studying itsstructural and functional characterization (Qian et al.2003). High-level production of the capsid protein (VP1)of human enterovirus 71 (EV71) was achieved in P. pastorisby codon optimization, and the recombinant VP1 couldinduce protective immune responses against EV71 infectionin mice (Wang et al. 2013).

To further characterize the purified protein, we performedelectrophoretic mobility shift assay to detect the DNA bindingactivity of the purified E1ΔGA (results not shown). Theyeast-expressed E1ΔGA showed comparable DNA bindingactivity as the E. coli-expressed EBNA1 (Duellman andBurgess 2006). The recombinant protein was then used toimmunize BALB/c mice to evaluate its immunogenicity atthe humoral and cellular levels. Serological test showed thatthe recombinant E1ΔGA induced high levels of E1ΔGA-specific antibodies in mice. Immunoblotting of B95-8 celllysates with the anti-E1ΔGA polyclonal antibodies demon-strated their reactivity with EBNA1, which further suggestedthe strong immunogenicity of the recombinant E1ΔGA. In aprevious study, a panel of monoclonal antibodies was gener-ated against the C terminus of EBNA1, and immunoblotanalysis demonstrated that the amino acids between aa 408and aa 498 were very immunogenic in mice (Chen et al.1999). The yeast-expressed E1ΔGA contains this immuno-genic domain, and thus the recombinant E1ΔGA could effi-ciently elicit high levels of EBNA1-specific antibodies inmice. It has been reported that EBNA1-specific CD4+ T cellsmediated primarily a Th1-type response (Paludan et al. 2002).The data presented here suggested that the immune responsesfollowing E1ΔGA immunization were provoked through theTh1-type route. Previously, three EBNA1 peptides were usedto immunize mice, and a slight antibody response was

observed in immunized mice against each EBNA1 peptide(Depil et al. 2007). Compared with these peptides, the recom-binant E1ΔGA could elicit higher levels of antibodies inimmunized mice (Fig. 5a). Recombinant EBNA1 proteinexpressed in insect cells could efficiently induce IgG anti-bodies to EBNA1 in mice after the first immunization (Yadavet al. 2011). In the present study, high levels of IgG antibodieswere also detected in E1ΔGA-immunized mice after theprimary immunization. The level of anti-E1ΔGA antibodyreached statistical significance compared with the controlmice, as measured by Student t-test, by week 4 (p<0.05;Fig. 5a). In a previous study, splenic cells from mice immu-nized with EBNA1 peptides showed a significant proliferativeresponse associated with IFN-γ secretion (eight- and ten-foldincrease) in vitro (Fu et al. 2004). In this study, we found thatE1ΔGA induced strong lymphoproliferative responses (p<0.01; Fig. 5c), and splenocytes from E1ΔGA-immunizedmice produced a higher level of IFN-γ (nine-fold increase)than the control mice when re-stimulated by E1ΔGA in vitro(p<0.01; Fig. 5d). The comparable levels of IFN-γ secretionas well as the similar magnitude of proliferative responsessuggested comparable levels of T lymphocyte stimulation bythese antigens. Furthermore, the recombinant E1ΔGA couldinduce T cell responses which might play a role in the controlof EBV-associated malignancies.

In conclusion, we have successfully expressed theE1ΔGA protein in P. pastoris with high levels and goodimmunogenicity. P. pastoris as an ideal expression systemfor the optimized E1ΔGA gene was confirmed in this work.The high-level expressed E1ΔGA yield reached210.53 mg/L. To the best of our knowledge, the achievedE1ΔGAyield is the highest ever reported. The procedure forthe production of E1ΔGA described in this study is cost-effective, manageable, and scalable. The yeast-expressedE1ΔGA elicited both humoral and cellular responses inmice, which demonstrated the strong immunogenicity ofthe recombinant protein. Further works are required tounderstand the protective efficiency and other aspects ofthe recombinant E1ΔGA. This study might have importantimplications for the development of an effective vaccineagainst EBV-associated tumors.

Acknowledgments This work was financially supported by theNational Basic Research Program of China (973 Program, no.2011CB504800).

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