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Expression and Characterization of HIV-1 Envelope Glycoprotein in Pichia Pastoris
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CHEM, RES. CHINESE UNIVERSITIES
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2008,24(3), 316-321 Article ID 1005-9040(2008)-03-316-06
=f' ScienceDirect
Expression and Characterization of HIV-1 Envelope Glycoprotein in Pichia Pastoris ZHAO Li-hui', W Xiang-hui2, JIANG Chun-lai2,WU Yong-ge2, SHEN Jia-cong3*and KONG Wei2* I . Changchun University of Science and Techinologv, Changchun 130022, I? R. China; 2. College of Life Science, 3. State Key Laboratory for Supramolecular Structure and Materials, Jilin University, Changchun 130012, I? R. China Abstract To obtain a sufficient amount of glycoprotein for further studying the structure and function of HIV-1 envelope glycoprotein, amplified and modified HIV-1 envelope glycoprotein gene which recombined subtypes(850 amino acids) from Guangxi in China was inserted into Pichiapastoris expression vector pP1CZaB; then the recombinant plasmid was transported into the yeast cells to induce the expression of Env protein with methanol. The results of SDS-PAGE and Western blot indicate that the envelope glycoprotein could be expressed in Pichia pustoris with productions of a 120000 glycoprotein and a 41000 glycoprotein, which showed satisfactory immunogenicity by indirect ELISA. Keywords Pichia pastoris; env gene; Glycoprotein;Epitope; Immunogenicity
I Introduction Since Human immunodeficiency virus(H1V) was discovered in 1985, it has spread over the world astonishingly and the number of patients infected with HIV increases day by day. Though several human efforts and economic resources are devoted to exploring medicines for the prevention and treatment of AIDS, there has been no vaccine or medicine that could prevent or cure AIDS completely by now. Therefore, it is important to develop reagents to detect AIDS. The entire HIV-1 envelope glycoprotein is the structural protein of HIV-1 virus. The molecular weight of its precursor is 160000(gp160), which has been processed into the external membrane protein gp120 and transmembrane protein gp41. The binding sites for V3 and CD4 in the complementary determining regions(CDR) of gp120 could stimulate the organism to produce crucial antigens for systemic humoral and cellular immune responses"]. Moreover, gp120 is highly glycosylated, with more than half of its molecular weight from glycosylated chains. All the 24 potential sites for N-glycosylation have been utilizedL2],and the glycoprotein might be modified by the
addition of 0-linked carbohydrate^'^]. Therefore, they might be much safer when applied clinically since the glycosylation pattern of the protein products in Pichia pustoris is similar to that of eukaryotest4]. Since 19703, more than 30 heterologous genes have been successfully expressed with the help of yeast systems, most of the proteins expressed by which were medical products and some of which have entered into the Since the yeast expression vector pPICZaB used here is a high-efficiently secretory expression vector with secreting signal sequence, the produced protein could be secreted into the fermentation broth. In addition, there is DNA sequence homologous to that of the yeast genome in the expression vector, so the target gene could be integrated into the yeast g e n ~ m e [ ~ , ~ ] . After analyzing and purifying the protein, we analyzed its immunogenicity, hoping for obtaining antigens which could detect all subtypes of HIV-1 antibodies.
2 Experimental 2.1
Materials Pichiu pustoris GS 115 and plasmid pPICZaB
*Corresponding author. E-mail: [email protected]; [email protected] Received October 30,2007; accepted December 21,2007. Supported by Jilin Basic Reesearch Program, China(No.20010710). Copyright Q 2008, Jilin University. Published by Elsevier Limited. All rights reserved.
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were purchased from Invitrogen Company, Ecscherichiu coli DH5a was kept at the laboratory. PGEM-T-easy vector, restriction endonuclease, T4 ligase and Tuq DNA polymerase were all purchased from TaKaRa Company. Regular reagents for yeast culture and the culture medium were prepared according to the manufacturer's instructions for yeast(re1eased by the Invitrogen Company).
2.2 Amplification of env Gene According to the full-length env gene sequence, two primers were designed(Primers were synthesized by Dalian Biological Engineering Corporation). The forward primer was S-GAATTCAGTCGACATGAGAGTGAGGGGGACA-3', and the backward primer was 5'-TCTAGACCATGGTTACTG?'TATTGCAAAGCTG-3'. A DNA fragment of interest, about 2586 bp in length, was amplified from the template of plasmid VR 101 2 - e ~with HIV-1 envelope glycoprotein encoding gene by PCR. After being recovered and purified from agarose gel, the PCR product was cloned into vector Pgem-Teasy.
2.3 Construction of Recombinant Plasmid pPICZaB-env Both pGEM-T-easy-env and pPICZaB were doubly digested by restriction endonucleases SUN Ncol. The yeast GS115 was prepared into the competent cells under the instructions of the Invitrogen Company. 80 pL of the competent cells and 5-10 pg of recombinant expression plasmid pPICZaB-em linearized by Sac1 were transferred into 0.2 cm electroporation cuvette, which were incubated for 5 minutes. Set the Bio-Rad Gene Pulser to deliver an electrical pulse of 1500 V ,25 pF, 200 a. Immediately add 1 mL of ice-cold 1 moVL sorbitol to the cuvette. The cell suspension was spread on the YPDS selective plates that were incubated for 2-3 days at 28-30 "C. Transformants were screened and identified for the successful transformation of recombinant plasmid pPICZaB-env. 2.4 Screening and Identification of His+ Mut' Transformants
Transformed colonies were placed on HMD and HMM selection plates correspondingly and the yeast strains grown well on each selection plate were screened. After a single colony was inoculated into a
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flask containing 5 mL of YPD cultures and incubated with vigorous shaking for 16-20 h at 28-30 "C, the cells were harvested by centrifugation. To suspend the cell pellet in 200 pL of lysis buffer were added 200 pL of glass beads of 0.5 mm in diameter and 200 pL of saturated phenol. After shaking and centrifugation, the genome DNA was precipitated from supernatant by 100% ethanol, digested by RNase, extracted by phenol: chloroform, and finally dissolved in 40 pL of TE buffer. Taking the extracted yeast genome as the template, PCR was performed with AOXl general primers on the yeast vector under the above-described PCR conditions. AOX 1 general primers are: 5'-GACTGGTTCCAATTGACAAGCG-3' and 3'-GCAAATGGCATTCTGACATCC-5'. 2.5 Induction of env Gene Expression
In 5 mL of YPD liquid culture was inoculated a single positive colony, overnight at 28-30 OC. The cells collected by centrifugation were resuspended in 100 mL of BMGY culture medium and incubated at 28-30 "C till the value of OD=2.0-6.0. The cells were collected and resuspended in 50 mL of BMMY culture till the value of OD=1.0 and incubated for 5 days at 25 "C, by adding methanol to a final concentration of 1% each day. The supernatant was collected by centrifugation, and the products in the Supernatants were analyzed by SDS-PAGE and Western blot. The primary antibody was HIV positive serum, and the secondary antibody was alkaline phosphatase conjugated goat anti-human IgG. NBTBCIP. 2.6 Purification and Characterization of Expressed Products
After the recombinant yeast was highly expressed-in BMMY culture, the supernatant was collected The molecular-sieve chromatographic His' column and 35% Sucrose density gradient centrifugation(26000g, 1.5 h) were used to purify Env protein. The expression of the protein was analyzed by SDS-PAGE and identified by Western blot. The content of the sugar was measured by colorimetric method using phenol-sulfuric acidtg1. 2.7 Detection of Immunogenicity
The purified antigen was diluted to a final concentration of 1 pg/mL in coating buffer(50 nmoVL of carbonate buffer, pH=9.6) to coat the polystyrene plate.
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After the plate was incubated for 2 h at 37 "C, 200 pL of blocking buffer(dried skim milk of 1% PBS) was added to each well and incubated for 2 h at 37 "C. Human serum sample was diluted to 1:1000(volume ratio) in blocking buffer, 100 pL of which was added to each well and incubated for 2 h at room temperature. Enzyme-linked rabbit anti-human secondary antibody was diluted to 1:5000(volume ratio) in blocking buffer, 100 pL of which was added to each well and incubated for 30 min at room temperature. A 100 mmol/L PBS solution(pH 6.0) of 10 mL, 100 pL of TMB stock solution and 15 pL of 30% H202 were mixed when used, and 100 pL of the mixed solution was added to each well. The color develop reaction was processed for 10 minutes and the absorbance readings were observed at a wavelength of 450 nm.
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white screening method. The plasmid of the white colonies is extracted and the plasmid T-easy-env is digested by double restriction endonucleases EcoRIl XbaI[Fig.l(A)]. DNA fragment of 2.5 kb was recovered from the agarose gel. Relatively, the vector pPICZaB was doubly digested by EcoRIIXbaI, and then recovered. The two were ligated by T4 DNA ligase with a certain proportion(env: pPICZaB=3: I), and then transformed into competent cells JM109. The right colonies were picked, the plasmid of which was doubly digested by EcoRIl'aI, which resulted in two DNA fragments of 3.6 and 2.5 kb[Fig.l(B)]. The results of PCR and DNA sequencing of the recombinant plasmid pPICZaB-env confrmed the success of recombinant plasmid construction.
2.8 Observed Result of Transmission Electron Microscopy
Protein sample of Env purified by Sucrose density gradient centrifugation was dissolved in 20 pL of 5 mmol/L Tris-HC1 phosphatic buffer. A drop of the suspension was dripped on the copper net, negatively stained with 2% uranyl acetate, and observed by means of transmission electron microscopy.
3 Results 3.1 Construction of Recombinant Plasmid pPICZaB-env The expression of heterologous gene in yeast is related to the selection of heterologous gene. Understanding of the preference to the codons in the expression system host is critical to analyze the criteria of heterologous gene expression at the translational level, providing proofs for altering heterologous genes or host cells. HIV- 1 envelope glycoprotein encoding gene containing rare DNA sequences, which could not be completely expressed in yeast, was modified and then inserted into T-easy vector. Simultaneously, two primers were designed (including restriction cleavage sites for EcoR I/Xba I). PCR was used to amplify the gene of the interest which was about 2580 bp, with the plasmid T-easy-env as the template. The sample of PCR product was loaded on the agarose gel of 1%. The result of gel electrophoresis suggests that the PCR product of the right size is obtained. Recovered and purified from the gel, the product of PCR was ligated into vector pGEM-T-easy, and identified by blue-
Fig. 1 Agarose gel electrophresis of T-easy-env(A) and pPICZaB-env cut by EcoR I and XbaI(B) (A) Lane 1: T-easy-env cut by SalI and NcoI(3010 bp, 2580 bp); lane 2: 1 kb plus DNA ladder; (B) lane I : 1 kb DNA plus ladder; lane 2: recombinant plasmid(6180 bp); lane 3: recombinant plasmid cut by EcoRI and xbaI(3600 bp, 2580 bp).
3.2 Screening of Phenotypes of His'Mut' Identification of Positive Colonies
and
Homologous recombination in yeast is the dificulty of the recombinant plasmid construction. The homologous recombinant rate of unlinearized circular plasmid is too low, so the recombinant transfer vector has to be linearized, digested by specific restriction endonuclease. The aim was to prevent the functional region of the plasmid from breaking out and inactivating the expression of the target gene when random recombination occurred, so that homologous recombination would take place in the directed way. pPICZaB-env and pPICZaB linearized by Sac1 were electroporated into the yeast GS115. The transformed cells was incubated for 2-3 d at 28-30 "C till the single colonies grew to an appropriate size. The right phenotypes were screened on a HMD and HMM selection plate. Select 10 transformants with the phenotype of Muti, and their genomes were amplified to
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identify the positive recombinants by PCR with general primers on the yeast chromosome. The result of the PCR indicates that not all the transformants with the phenotype of Mut' were the positive recombinants. The positive recombination rate was 90% in all the selected transformants. In positive recombinants, env gene was integrated into the genome of Pichia pastoris by double crossover. The result of the PCR of the recombinant yeast genome containing pPICZaB-env reveals two DNA fragments of 3 170 bp(2580 bp+590 bp) and 2200 bp in length, which suggests that the aim gene env had been integrated into the yeast genome. Gene AOXl on the yeast genome was 2200 bp in length, and the general fragment of AOXl on the yeast vector was 590 bp. While the PCR results of recombinant yeast containing pPICZaB reveal two DNA fragments of 2200 bp and 590 bp, which indicates that no homologous recombination took place in the negative recombinants(Fig.2).
Fig.2 PCR analysis of Bchiapastods clones Lane 1 1 kb DNA ladder; lane 2. control clone(2200 bp, 590 bp), lane 3 clone carrying aim gene(2200 bp, 3170 bp).
3.3 Induction of Expression of Recombinant Yeast The single colony was inoculated in the culture at 28-30 "C via 85% aeration in a shaking incubator, by adding 100% methanol to a final concentration of 0.5%, 1% or 1.5%, respectively, and supplement was carried out once every 24 h to maintain the induction. Samples were taken at various time points during the induction period(0, 6, 12, 24, 36, 48, 60, 72, 84 and 96 h), to carry out SDS-PAGE and Western blot analyses. The results indicate that an induction time of 72 h and a final methanol concentration of 1% were the optimum inducing conditions with the maximum expression of the aim gene. Western blot analysis of the recombinant yeast shows that two obviously colored bands were visualized, which were determined to be specific protein bands of 120 and 41 kDa approximately(Fig.3). Pichia pastoris could take advantage
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of methanol as the exclusive carbon source to grow and reproduce fast. Meanwhile, as the inducer, the expression amount of heterologous genes might be positively related to the concentration of the methanol.
Fig.3 SDS-PAGEand Westernblot analysis of protein Env (A) Lane 1 protein molecular weight marker, lane 2 induced supernatant of GSllS/pPICZaB-env strain(72 h), lane 3 induced supernatant of GSIIS/pPICZaB strain(72 b,control), (B) lane I protein induced supernatant of molecularweight marker, lane 2 GSIIS/pPICZaB strain(72 h, control), lane 3 induced supernatant of GSI ISlpPICZaB-env strain(72 h).
Incubating in shaking flasks, the content of the expressed protein Env was positively related to the methanol concentration when it ranged from 0.5% to 2%. Later, its content decreased with the methanol concentration of above 3%. So inducing by 1% methanol, the content of the expressed product increased with the induction time extending. Till 72 h, it approached the peak, and the content of the protein of interest accounted for 19% of the total protein content. When induction time extended to 96 h, the content of the protein of interest did not increase any more, but decreased slightly instead. Aeration is one of the most important factors influencing the expression of recombinant cells. More than 85% aeration was necessary to maintain the high-level expression of the protein of interest. Induced by 1% methanol, the results indicate that with the increasing agitation speed, the productivity of protein Env was highly raised. When the pH approached 6.0-6.5, the expression of protein Env reached the highest point. Yeast could grow in a wide range of pH from 3 to 8. Setting the pH value during the induction in a certain range not only benefits the internal physiological condition of the yeast cells, but also could inhibit the activity of some protease to reduce the degradation of the protein of interest. To obtain the high-level expression, it is necessary to optimize the inducing conditions for massive expression. Therefore, €? pastoris is incubated in shaking flasks to optimize the expressing conditions for the secretion of the expressed HIV-1 protein Env, to expand the production. The positive clones identi-
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CHEM. RES. CHINESE UNIVERSITIES
fied by PCR were selected to be induced, with the yeast strain GS 115 integrated with the empty expression vector pPICZaB as the negative controls, The supernatants were picked up to perform SDS-PAGE and Western blot analysis as regular.
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gated goat anti-human IgG as the secondary antibody, Western blot was carried out to characterize its immunogenicity(Fig.5).
3.4 Purification and Characterization of Expressed Protein
I
The yeast strains which could highly express HIV-1 Env antigen fiagment were constructed using l? pastoris expression system. The expressed heterologous protein was secreted out of the yeast cells, which brought a great convenience for its purification. Additionally, the expressed fusion protein contained an exposed His-6 sequence at the amino-terminus. Because, polyhistidine could attach with significant affinities to transition metals(e.g. Nit, Zn2+, Cu2+, et d.), charged metal ligand affinity chromatography could be used for fast purification. Metal chelate chromatography is a protein isolation technique and a useful tool for the purification of the target proteins with polyhistidine in the expression system. Accordingly, the aim proteins were purified using Nisepharose 4B as metal chelate matrix. The purified protein was detected by staining with Coomassie Brilliant Blue. With HIV-1 positive serum of affected patients as the primary antibody, and alkaline phosphatase conjugated goat anti-huaman IgG as the secondary antibody, Western blot was carried out to characterize its immunogenicity(Fig.4).
Fig.4 SDS-PAGE and Western blot analyses of purified Env protein (A) Lane 1: protein molecular weight marker; lane 2: purfied Env
protein; lane 3: induced supernatant of GSI 1SlpPICZaB-env strain(72 h, control); (B) lane 1: protein molecular weight marker; lane 2: induced supernatant of GSI IS/pPICZaB strain(first antibody HIV-I serum, 72 h, control); lane 3: supernatant of purified Env protein(first antibody HIV-I serum).
The supernatants were sedimentated through a 35% cushion. The sediment was analyzed by SDS-PAGE, and two protein bands were visualized. With HIV-1 positive serum of affected patients as the primary antibody, and alkaline phosphatase conju-
Fig.5 SDS-PAGE and westernblot analyses of purfied Env protein (A) Lane 1: induced
supernatant of GSl ISlpPICZaB-env strain(72 h); lane 2: protein molecular weight marker; lane 3: supernatant of GS11S/pPICZaB-env strain(72 h) by ultracentrifugation in a sucrose gradient 35%; (B) lane 1: protein marker; lane 2. supernatant of GSl 15lpPICZaB strain(72 h) by ultracentrifugation in a sucrose gradient 35%; lane 3: supernatant of GSIlSlpPICZaB-env strain(72 h) by ultracentrifugation in a sucrose gradient 35%.
3.5 Measurement of Sugar Content of Recombinant Protein Env The glycosylated modification of HIV- 1 envelope glycoprotein is critical to maintain its biological activity and immunogenicity. While, both prokaryotic expression and the internal expression in l? pastoris could not realize the post-translationally glycosylated modification of the products. Thus, HIV-1 glycoprotein, Z? pastoris is a fairly ideal secretion expression system. The expressing HIV envelope glycoprotein in l? pastoris lies in that l? pastoris expression system could modifL the Asn residue secreted in the AsnXSer/Thr of heterologous proteins by N-linked glycoprotein, which is consistent with natural HIV envelope glycoprotein"O1.The internal expression in l? pustoris could realize the post-translational glycosylation of the produced protein. With the signal sequence of the yeast on the expression vector, the protein of interest could be secreted outside the cells, which was beneficial to purification. Colorimetric method was used to measure the sugar content in glycoprotein(pheno1-sulfuric acid method)[']. The result indicates the sugar content of the expressed envelope glycoprotein that accounted for 32% of the total glycoprotein, indicating that the expressed product was glycoprotein definitely. To c o n f m the production of HIV-1 glycoprotein Env in this system, morphological test was used. The sample of protein Env purified by density gradient
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centrifugation was dissolved in 20 pL of 5 mmol/L Tris-HC1buffer. A drop of the solution was dripped on the copper net, negatively stained with uranyl acetate, and virus-like particles were viewed under a transmission electron microscope. The size of the particles was calculated to be in a range of 60-150 nm in diameter(Fig.6 ) .
Fig.6
Structure of purified protein in Env electron microscopy(60-150 nm)
3.6 Immunogenicity of Purified Protein The purified recombinant protein was used as an antigen to coat the ELISA plate(l00 ng per well). Indirect ELISA was performed to detect the HIV positive sera and negative sera provided by National Institute for the Control of Pharmaceutical Products(the samples were collected from the population with sensitivity and at high risk throughout the country, and defined as positive and negative serum samples after screening). The results of ELISA are shown in Fig.7.
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coli and it is inferred that the toxic activity affects the cell's growing In addition, too many rare codons in Escherichia coli result in no expression or partial degradation of interest pr~tein"~'. Gp160 is a highly glycosylated protein, with more than 20 potential sites for glycosylation in gp120; so yeast expression system should be selected to express HIV-I envelope antigen. Since the encoding sequence of HIV-1 envelope protein could be partially expre~sed"~], its encoding sequence so that the entire Env protein could be expressed in yeast. The studies indicate that HIV- 1 envelope glycoprotein assembled"51 and the aggregations are obtained by Sucrose density gradient Centrifugation. Analysis of the molecular weight of the protein shows that the sugar content in glycoprotein accounts for 35% approximately. Transmission electron microscopy was applied to observing the structure of HIV- 1 envelope glycoprotein Env collected by Sucrose density gradient centrifugation, which revealed that it assembled or formed virus-like particles. References [I] Bolognesi D., J Acquired Immune Defi. Syndr., 1990,3, 390 [2] Leonard C. K., Spellman M. W., Riddle L., e f al., J. Biol. Chem., 1990,265(18), 10373 [3] Bemstein H. B., Tucker S. P., Hunter E., e f al., J. Mrol., 1994,463 [4] Yang Liming, Zhang Hui-&an, Zhang Si-liang, et a/., Pmg. Biochem. Biophys., 2000,27(2), 15 1 [ 5 ] Li Jing, Zhao Xiao-xiang, Sha Chang-qing, er al., Progress in Bio-
fechnology, 1999, f9(2), 17
1.5
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[6] Nie Dong-song, Liang Song-ping, Li Min., Journal of Jishou University, Natural Science Edition,2001,22(3), 41
-
[7] Li Zi-jian, Jin Ning-yi, Jiang Wen-zheng, et al., Jaurnal of Jilin Universziy, Medicine Edition, 2003,2(29), 135
I
3
4
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[8] Zhang Ying-Jiu, Jin Ning-Yi, Wang Hong-wei, et al., Actu Biochimzcu et Biopbsica Sinica, 2001,33(4), 405
n 0
[9] Dubois M., Gilles K. A, Hamilton J. K., et al., Anal. Chem., 1956, 28(3), 350 1
2
3
4
Sample number
Fig.7
Evaluation of anti-HIV of purified recombinant Env protein
1. Negative control; 2. positive control; 3. purified protein Env expressing in P. pastoris by ultracentrifugation in a sucrose gradient 35%; 4. purified Env protein expressing in P pasforrs hy Ni-sepharose 4B.
4 Discussion Large quantities of experiments demonstrate that it is difficult to express HIV-1 gp160 in Escherichia
[ 101 Grinna L. S., Tschopo J. F., Yeast, 1989,5(2), 107 [ 1I ]
Sohn M. J., Lee M. E., Park H. S., et al., J Biotechnol., 1996,45(3), 211
[I21 Yuan Yu-hua, Bi Chang-hao, Li Ju, et al., Chinese Science Bulletin, 2004,49(2), 173 [I31 Zhao Li-hui,Yu Xiang-hui, Jiang Chun-lai, et a/.,J. B i d . Tech. Chin., 2007,3,457 [ 141 Jiang Jing, Zhao Li-hui, Yu Xiang-hui, et al., J. Biol. Chem., 2005, 12(26), 12259 1151 Doan L. X., Li Min, Chen Chang-yi, e f al., Rev. Med. Mrol., 2005, 15,
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2008,24(3), 316-321 Article ID 1005-9040(2008)-03-316-06
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Expression and Characterization of HIV-1 Envelope Glycoprotein in Pichia Pastoris ZHAO Li-hui', W Xiang-hui2, JIANG Chun-lai2,WU Yong-ge2, SHEN Jia-cong3*and KONG Wei2* I . Changchun University of Science and Techinologv, Changchun 130022, I? R. China; 2. College of Life Science, 3. State Key Laboratory for Supramolecular Structure and Materials, Jilin University, Changchun 130012, I? R. China Abstract To obtain a sufficient amount of glycoprotein for further studying the structure and function of HIV-1 envelope glycoprotein, amplified and modified HIV-1 envelope glycoprotein gene which recombined subtypes(850 amino acids) from Guangxi in China was inserted into Pichiapastoris expression vector pP1CZaB; then the recombinant plasmid was transported into the yeast cells to induce the expression of Env protein with methanol. The results of SDS-PAGE and Western blot indicate that the envelope glycoprotein could be expressed in Pichia pustoris with productions of a 120000 glycoprotein and a 41000 glycoprotein, which showed satisfactory immunogenicity by indirect ELISA. Keywords Pichia pastoris; env gene; Glycoprotein;Epitope; Immunogenicity
I Introduction Since Human immunodeficiency virus(H1V) was discovered in 1985, it has spread over the world astonishingly and the number of patients infected with HIV increases day by day. Though several human efforts and economic resources are devoted to exploring medicines for the prevention and treatment of AIDS, there has been no vaccine or medicine that could prevent or cure AIDS completely by now. Therefore, it is important to develop reagents to detect AIDS. The entire HIV-1 envelope glycoprotein is the structural protein of HIV-1 virus. The molecular weight of its precursor is 160000(gp160), which has been processed into the external membrane protein gp120 and transmembrane protein gp41. The binding sites for V3 and CD4 in the complementary determining regions(CDR) of gp120 could stimulate the organism to produce crucial antigens for systemic humoral and cellular immune responses"]. Moreover, gp120 is highly glycosylated, with more than half of its molecular weight from glycosylated chains. All the 24 potential sites for N-glycosylation have been utilizedL2],and the glycoprotein might be modified by the
addition of 0-linked carbohydrate^'^]. Therefore, they might be much safer when applied clinically since the glycosylation pattern of the protein products in Pichia pustoris is similar to that of eukaryotest4]. Since 19703, more than 30 heterologous genes have been successfully expressed with the help of yeast systems, most of the proteins expressed by which were medical products and some of which have entered into the Since the yeast expression vector pPICZaB used here is a high-efficiently secretory expression vector with secreting signal sequence, the produced protein could be secreted into the fermentation broth. In addition, there is DNA sequence homologous to that of the yeast genome in the expression vector, so the target gene could be integrated into the yeast g e n ~ m e [ ~ , ~ ] . After analyzing and purifying the protein, we analyzed its immunogenicity, hoping for obtaining antigens which could detect all subtypes of HIV-1 antibodies.
2 Experimental 2.1
Materials Pichiu pustoris GS 115 and plasmid pPICZaB
*Corresponding author. E-mail: [email protected]; [email protected] Received October 30,2007; accepted December 21,2007. Supported by Jilin Basic Reesearch Program, China(No.20010710). Copyright Q 2008, Jilin University. Published by Elsevier Limited. All rights reserved.
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were purchased from Invitrogen Company, Ecscherichiu coli DH5a was kept at the laboratory. PGEM-T-easy vector, restriction endonuclease, T4 ligase and Tuq DNA polymerase were all purchased from TaKaRa Company. Regular reagents for yeast culture and the culture medium were prepared according to the manufacturer's instructions for yeast(re1eased by the Invitrogen Company).
2.2 Amplification of env Gene According to the full-length env gene sequence, two primers were designed(Primers were synthesized by Dalian Biological Engineering Corporation). The forward primer was S-GAATTCAGTCGACATGAGAGTGAGGGGGACA-3', and the backward primer was 5'-TCTAGACCATGGTTACTG?'TATTGCAAAGCTG-3'. A DNA fragment of interest, about 2586 bp in length, was amplified from the template of plasmid VR 101 2 - e ~with HIV-1 envelope glycoprotein encoding gene by PCR. After being recovered and purified from agarose gel, the PCR product was cloned into vector Pgem-Teasy.
2.3 Construction of Recombinant Plasmid pPICZaB-env Both pGEM-T-easy-env and pPICZaB were doubly digested by restriction endonucleases SUN Ncol. The yeast GS115 was prepared into the competent cells under the instructions of the Invitrogen Company. 80 pL of the competent cells and 5-10 pg of recombinant expression plasmid pPICZaB-em linearized by Sac1 were transferred into 0.2 cm electroporation cuvette, which were incubated for 5 minutes. Set the Bio-Rad Gene Pulser to deliver an electrical pulse of 1500 V ,25 pF, 200 a. Immediately add 1 mL of ice-cold 1 moVL sorbitol to the cuvette. The cell suspension was spread on the YPDS selective plates that were incubated for 2-3 days at 28-30 "C. Transformants were screened and identified for the successful transformation of recombinant plasmid pPICZaB-env. 2.4 Screening and Identification of His+ Mut' Transformants
Transformed colonies were placed on HMD and HMM selection plates correspondingly and the yeast strains grown well on each selection plate were screened. After a single colony was inoculated into a
317
flask containing 5 mL of YPD cultures and incubated with vigorous shaking for 16-20 h at 28-30 "C, the cells were harvested by centrifugation. To suspend the cell pellet in 200 pL of lysis buffer were added 200 pL of glass beads of 0.5 mm in diameter and 200 pL of saturated phenol. After shaking and centrifugation, the genome DNA was precipitated from supernatant by 100% ethanol, digested by RNase, extracted by phenol: chloroform, and finally dissolved in 40 pL of TE buffer. Taking the extracted yeast genome as the template, PCR was performed with AOXl general primers on the yeast vector under the above-described PCR conditions. AOX 1 general primers are: 5'-GACTGGTTCCAATTGACAAGCG-3' and 3'-GCAAATGGCATTCTGACATCC-5'. 2.5 Induction of env Gene Expression
In 5 mL of YPD liquid culture was inoculated a single positive colony, overnight at 28-30 OC. The cells collected by centrifugation were resuspended in 100 mL of BMGY culture medium and incubated at 28-30 "C till the value of OD=2.0-6.0. The cells were collected and resuspended in 50 mL of BMMY culture till the value of OD=1.0 and incubated for 5 days at 25 "C, by adding methanol to a final concentration of 1% each day. The supernatant was collected by centrifugation, and the products in the Supernatants were analyzed by SDS-PAGE and Western blot. The primary antibody was HIV positive serum, and the secondary antibody was alkaline phosphatase conjugated goat anti-human IgG. NBTBCIP. 2.6 Purification and Characterization of Expressed Products
After the recombinant yeast was highly expressed-in BMMY culture, the supernatant was collected The molecular-sieve chromatographic His' column and 35% Sucrose density gradient centrifugation(26000g, 1.5 h) were used to purify Env protein. The expression of the protein was analyzed by SDS-PAGE and identified by Western blot. The content of the sugar was measured by colorimetric method using phenol-sulfuric acidtg1. 2.7 Detection of Immunogenicity
The purified antigen was diluted to a final concentration of 1 pg/mL in coating buffer(50 nmoVL of carbonate buffer, pH=9.6) to coat the polystyrene plate.
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After the plate was incubated for 2 h at 37 "C, 200 pL of blocking buffer(dried skim milk of 1% PBS) was added to each well and incubated for 2 h at 37 "C. Human serum sample was diluted to 1:1000(volume ratio) in blocking buffer, 100 pL of which was added to each well and incubated for 2 h at room temperature. Enzyme-linked rabbit anti-human secondary antibody was diluted to 1:5000(volume ratio) in blocking buffer, 100 pL of which was added to each well and incubated for 30 min at room temperature. A 100 mmol/L PBS solution(pH 6.0) of 10 mL, 100 pL of TMB stock solution and 15 pL of 30% H202 were mixed when used, and 100 pL of the mixed solution was added to each well. The color develop reaction was processed for 10 minutes and the absorbance readings were observed at a wavelength of 450 nm.
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white screening method. The plasmid of the white colonies is extracted and the plasmid T-easy-env is digested by double restriction endonucleases EcoRIl XbaI[Fig.l(A)]. DNA fragment of 2.5 kb was recovered from the agarose gel. Relatively, the vector pPICZaB was doubly digested by EcoRIIXbaI, and then recovered. The two were ligated by T4 DNA ligase with a certain proportion(env: pPICZaB=3: I), and then transformed into competent cells JM109. The right colonies were picked, the plasmid of which was doubly digested by EcoRIl'aI, which resulted in two DNA fragments of 3.6 and 2.5 kb[Fig.l(B)]. The results of PCR and DNA sequencing of the recombinant plasmid pPICZaB-env confrmed the success of recombinant plasmid construction.
2.8 Observed Result of Transmission Electron Microscopy
Protein sample of Env purified by Sucrose density gradient centrifugation was dissolved in 20 pL of 5 mmol/L Tris-HC1 phosphatic buffer. A drop of the suspension was dripped on the copper net, negatively stained with 2% uranyl acetate, and observed by means of transmission electron microscopy.
3 Results 3.1 Construction of Recombinant Plasmid pPICZaB-env The expression of heterologous gene in yeast is related to the selection of heterologous gene. Understanding of the preference to the codons in the expression system host is critical to analyze the criteria of heterologous gene expression at the translational level, providing proofs for altering heterologous genes or host cells. HIV- 1 envelope glycoprotein encoding gene containing rare DNA sequences, which could not be completely expressed in yeast, was modified and then inserted into T-easy vector. Simultaneously, two primers were designed (including restriction cleavage sites for EcoR I/Xba I). PCR was used to amplify the gene of the interest which was about 2580 bp, with the plasmid T-easy-env as the template. The sample of PCR product was loaded on the agarose gel of 1%. The result of gel electrophoresis suggests that the PCR product of the right size is obtained. Recovered and purified from the gel, the product of PCR was ligated into vector pGEM-T-easy, and identified by blue-
Fig. 1 Agarose gel electrophresis of T-easy-env(A) and pPICZaB-env cut by EcoR I and XbaI(B) (A) Lane 1: T-easy-env cut by SalI and NcoI(3010 bp, 2580 bp); lane 2: 1 kb plus DNA ladder; (B) lane I : 1 kb DNA plus ladder; lane 2: recombinant plasmid(6180 bp); lane 3: recombinant plasmid cut by EcoRI and xbaI(3600 bp, 2580 bp).
3.2 Screening of Phenotypes of His'Mut' Identification of Positive Colonies
and
Homologous recombination in yeast is the dificulty of the recombinant plasmid construction. The homologous recombinant rate of unlinearized circular plasmid is too low, so the recombinant transfer vector has to be linearized, digested by specific restriction endonuclease. The aim was to prevent the functional region of the plasmid from breaking out and inactivating the expression of the target gene when random recombination occurred, so that homologous recombination would take place in the directed way. pPICZaB-env and pPICZaB linearized by Sac1 were electroporated into the yeast GS115. The transformed cells was incubated for 2-3 d at 28-30 "C till the single colonies grew to an appropriate size. The right phenotypes were screened on a HMD and HMM selection plate. Select 10 transformants with the phenotype of Muti, and their genomes were amplified to
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identify the positive recombinants by PCR with general primers on the yeast chromosome. The result of the PCR indicates that not all the transformants with the phenotype of Mut' were the positive recombinants. The positive recombination rate was 90% in all the selected transformants. In positive recombinants, env gene was integrated into the genome of Pichia pastoris by double crossover. The result of the PCR of the recombinant yeast genome containing pPICZaB-env reveals two DNA fragments of 3 170 bp(2580 bp+590 bp) and 2200 bp in length, which suggests that the aim gene env had been integrated into the yeast genome. Gene AOXl on the yeast genome was 2200 bp in length, and the general fragment of AOXl on the yeast vector was 590 bp. While the PCR results of recombinant yeast containing pPICZaB reveal two DNA fragments of 2200 bp and 590 bp, which indicates that no homologous recombination took place in the negative recombinants(Fig.2).
Fig.2 PCR analysis of Bchiapastods clones Lane 1 1 kb DNA ladder; lane 2. control clone(2200 bp, 590 bp), lane 3 clone carrying aim gene(2200 bp, 3170 bp).
3.3 Induction of Expression of Recombinant Yeast The single colony was inoculated in the culture at 28-30 "C via 85% aeration in a shaking incubator, by adding 100% methanol to a final concentration of 0.5%, 1% or 1.5%, respectively, and supplement was carried out once every 24 h to maintain the induction. Samples were taken at various time points during the induction period(0, 6, 12, 24, 36, 48, 60, 72, 84 and 96 h), to carry out SDS-PAGE and Western blot analyses. The results indicate that an induction time of 72 h and a final methanol concentration of 1% were the optimum inducing conditions with the maximum expression of the aim gene. Western blot analysis of the recombinant yeast shows that two obviously colored bands were visualized, which were determined to be specific protein bands of 120 and 41 kDa approximately(Fig.3). Pichia pastoris could take advantage
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of methanol as the exclusive carbon source to grow and reproduce fast. Meanwhile, as the inducer, the expression amount of heterologous genes might be positively related to the concentration of the methanol.
Fig.3 SDS-PAGEand Westernblot analysis of protein Env (A) Lane 1 protein molecular weight marker, lane 2 induced supernatant of GSllS/pPICZaB-env strain(72 h), lane 3 induced supernatant of GSIIS/pPICZaB strain(72 b,control), (B) lane I protein induced supernatant of molecularweight marker, lane 2 GSIIS/pPICZaB strain(72 h, control), lane 3 induced supernatant of GSI ISlpPICZaB-env strain(72 h).
Incubating in shaking flasks, the content of the expressed protein Env was positively related to the methanol concentration when it ranged from 0.5% to 2%. Later, its content decreased with the methanol concentration of above 3%. So inducing by 1% methanol, the content of the expressed product increased with the induction time extending. Till 72 h, it approached the peak, and the content of the protein of interest accounted for 19% of the total protein content. When induction time extended to 96 h, the content of the protein of interest did not increase any more, but decreased slightly instead. Aeration is one of the most important factors influencing the expression of recombinant cells. More than 85% aeration was necessary to maintain the high-level expression of the protein of interest. Induced by 1% methanol, the results indicate that with the increasing agitation speed, the productivity of protein Env was highly raised. When the pH approached 6.0-6.5, the expression of protein Env reached the highest point. Yeast could grow in a wide range of pH from 3 to 8. Setting the pH value during the induction in a certain range not only benefits the internal physiological condition of the yeast cells, but also could inhibit the activity of some protease to reduce the degradation of the protein of interest. To obtain the high-level expression, it is necessary to optimize the inducing conditions for massive expression. Therefore, €? pastoris is incubated in shaking flasks to optimize the expressing conditions for the secretion of the expressed HIV-1 protein Env, to expand the production. The positive clones identi-
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CHEM. RES. CHINESE UNIVERSITIES
fied by PCR were selected to be induced, with the yeast strain GS 115 integrated with the empty expression vector pPICZaB as the negative controls, The supernatants were picked up to perform SDS-PAGE and Western blot analysis as regular.
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gated goat anti-human IgG as the secondary antibody, Western blot was carried out to characterize its immunogenicity(Fig.5).
3.4 Purification and Characterization of Expressed Protein
I
The yeast strains which could highly express HIV-1 Env antigen fiagment were constructed using l? pastoris expression system. The expressed heterologous protein was secreted out of the yeast cells, which brought a great convenience for its purification. Additionally, the expressed fusion protein contained an exposed His-6 sequence at the amino-terminus. Because, polyhistidine could attach with significant affinities to transition metals(e.g. Nit, Zn2+, Cu2+, et d.), charged metal ligand affinity chromatography could be used for fast purification. Metal chelate chromatography is a protein isolation technique and a useful tool for the purification of the target proteins with polyhistidine in the expression system. Accordingly, the aim proteins were purified using Nisepharose 4B as metal chelate matrix. The purified protein was detected by staining with Coomassie Brilliant Blue. With HIV-1 positive serum of affected patients as the primary antibody, and alkaline phosphatase conjugated goat anti-huaman IgG as the secondary antibody, Western blot was carried out to characterize its immunogenicity(Fig.4).
Fig.4 SDS-PAGE and Western blot analyses of purified Env protein (A) Lane 1: protein molecular weight marker; lane 2: purfied Env
protein; lane 3: induced supernatant of GSI 1SlpPICZaB-env strain(72 h, control); (B) lane 1: protein molecular weight marker; lane 2: induced supernatant of GSI IS/pPICZaB strain(first antibody HIV-I serum, 72 h, control); lane 3: supernatant of purified Env protein(first antibody HIV-I serum).
The supernatants were sedimentated through a 35% cushion. The sediment was analyzed by SDS-PAGE, and two protein bands were visualized. With HIV-1 positive serum of affected patients as the primary antibody, and alkaline phosphatase conju-
Fig.5 SDS-PAGE and westernblot analyses of purfied Env protein (A) Lane 1: induced
supernatant of GSl ISlpPICZaB-env strain(72 h); lane 2: protein molecular weight marker; lane 3: supernatant of GS11S/pPICZaB-env strain(72 h) by ultracentrifugation in a sucrose gradient 35%; (B) lane 1: protein marker; lane 2. supernatant of GSl 15lpPICZaB strain(72 h) by ultracentrifugation in a sucrose gradient 35%; lane 3: supernatant of GSIlSlpPICZaB-env strain(72 h) by ultracentrifugation in a sucrose gradient 35%.
3.5 Measurement of Sugar Content of Recombinant Protein Env The glycosylated modification of HIV- 1 envelope glycoprotein is critical to maintain its biological activity and immunogenicity. While, both prokaryotic expression and the internal expression in l? pastoris could not realize the post-translationally glycosylated modification of the products. Thus, HIV-1 glycoprotein, Z? pastoris is a fairly ideal secretion expression system. The expressing HIV envelope glycoprotein in l? pastoris lies in that l? pastoris expression system could modifL the Asn residue secreted in the AsnXSer/Thr of heterologous proteins by N-linked glycoprotein, which is consistent with natural HIV envelope glycoprotein"O1.The internal expression in l? pustoris could realize the post-translational glycosylation of the produced protein. With the signal sequence of the yeast on the expression vector, the protein of interest could be secreted outside the cells, which was beneficial to purification. Colorimetric method was used to measure the sugar content in glycoprotein(pheno1-sulfuric acid method)[']. The result indicates the sugar content of the expressed envelope glycoprotein that accounted for 32% of the total glycoprotein, indicating that the expressed product was glycoprotein definitely. To c o n f m the production of HIV-1 glycoprotein Env in this system, morphological test was used. The sample of protein Env purified by density gradient
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centrifugation was dissolved in 20 pL of 5 mmol/L Tris-HC1buffer. A drop of the solution was dripped on the copper net, negatively stained with uranyl acetate, and virus-like particles were viewed under a transmission electron microscope. The size of the particles was calculated to be in a range of 60-150 nm in diameter(Fig.6 ) .
Fig.6
Structure of purified protein in Env electron microscopy(60-150 nm)
3.6 Immunogenicity of Purified Protein The purified recombinant protein was used as an antigen to coat the ELISA plate(l00 ng per well). Indirect ELISA was performed to detect the HIV positive sera and negative sera provided by National Institute for the Control of Pharmaceutical Products(the samples were collected from the population with sensitivity and at high risk throughout the country, and defined as positive and negative serum samples after screening). The results of ELISA are shown in Fig.7.
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coli and it is inferred that the toxic activity affects the cell's growing In addition, too many rare codons in Escherichia coli result in no expression or partial degradation of interest pr~tein"~'. Gp160 is a highly glycosylated protein, with more than 20 potential sites for glycosylation in gp120; so yeast expression system should be selected to express HIV-I envelope antigen. Since the encoding sequence of HIV-1 envelope protein could be partially expre~sed"~], its encoding sequence so that the entire Env protein could be expressed in yeast. The studies indicate that HIV- 1 envelope glycoprotein assembled"51 and the aggregations are obtained by Sucrose density gradient Centrifugation. Analysis of the molecular weight of the protein shows that the sugar content in glycoprotein accounts for 35% approximately. Transmission electron microscopy was applied to observing the structure of HIV- 1 envelope glycoprotein Env collected by Sucrose density gradient centrifugation, which revealed that it assembled or formed virus-like particles. References [I] Bolognesi D., J Acquired Immune Defi. Syndr., 1990,3, 390 [2] Leonard C. K., Spellman M. W., Riddle L., e f al., J. Biol. Chem., 1990,265(18), 10373 [3] Bemstein H. B., Tucker S. P., Hunter E., e f al., J. Mrol., 1994,463 [4] Yang Liming, Zhang Hui-&an, Zhang Si-liang, et a/., Pmg. Biochem. Biophys., 2000,27(2), 15 1 [ 5 ] Li Jing, Zhao Xiao-xiang, Sha Chang-qing, er al., Progress in Bio-
fechnology, 1999, f9(2), 17
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[6] Nie Dong-song, Liang Song-ping, Li Min., Journal of Jishou University, Natural Science Edition,2001,22(3), 41
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[7] Li Zi-jian, Jin Ning-yi, Jiang Wen-zheng, et al., Jaurnal of Jilin Universziy, Medicine Edition, 2003,2(29), 135
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[8] Zhang Ying-Jiu, Jin Ning-Yi, Wang Hong-wei, et al., Actu Biochimzcu et Biopbsica Sinica, 2001,33(4), 405
n 0
[9] Dubois M., Gilles K. A, Hamilton J. K., et al., Anal. Chem., 1956, 28(3), 350 1
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Sample number
Fig.7
Evaluation of anti-HIV of purified recombinant Env protein
1. Negative control; 2. positive control; 3. purified protein Env expressing in P. pastoris by ultracentrifugation in a sucrose gradient 35%; 4. purified Env protein expressing in P pasforrs hy Ni-sepharose 4B.
4 Discussion Large quantities of experiments demonstrate that it is difficult to express HIV-1 gp160 in Escherichia
[ 101 Grinna L. S., Tschopo J. F., Yeast, 1989,5(2), 107 [ 1I ]
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[I21 Yuan Yu-hua, Bi Chang-hao, Li Ju, et al., Chinese Science Bulletin, 2004,49(2), 173 [I31 Zhao Li-hui,Yu Xiang-hui, Jiang Chun-lai, et a/.,J. B i d . Tech. Chin., 2007,3,457 [ 141 Jiang Jing, Zhao Li-hui, Yu Xiang-hui, et al., J. Biol. Chem., 2005, 12(26), 12259 1151 Doan L. X., Li Min, Chen Chang-yi, e f al., Rev. Med. Mrol., 2005, 15,
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