key: cord-0005382-2xpkvtrb
authors: Qian, Ping; Li, Xiangmin; Tong, Guangzhi; Chen, Huanchun
title: High-level Expression of the ORF6 Gene of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in Pichia pastoris
date: 2003
journal: Virus Genes
DOI: 10.1023/a:1025784712265
sha: ec0169d46b60dc120f431e11e71f0a908890a148
doc_id: 5382
cord_uid: 2xpkvtrb

High-level expression of the ORF6 gene of porcine reproductive and respiratory syndrome virus (PRRSV) has been proved very difficult. In this work, we cloned and sequenced the ORF6 gene of PRRSV and found that it could not be expressed in Pichia pastoris strain GS115. Then, the ORF6 gene was modified and synthesized based on the codon bias, poly (A) signal of yeast expression system and secondary structure of 5′-end mRNA of foreign gene. The modified gene was inserted into the yeast expression vector pPICZαA, induced and expressed by the same methods. The recombinant protein with a molecular mass of approximately 23 kDa was screened by SDS-PAGE and identified by Western blot with convalescent sera of animals infected with CH-1a strain of PRRSV. The results indicated that it was similar to the native protein. The expression level of the recombinant protein could attain 2.0 g/L. In the meanwhile, the optimal conditions for expression were determined. It provides an additional means for studying the structural and functional characteristics of PRRSV ORF6 gene.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the Arteriviridae family which includes lactate dehydrogenase-elevating virus (LDV) of mice, equine arteritis virus (EAV), and simian hemorrhagic fever virus (SHFV) [1, 3, 5, 18] . PRRSV is a relatively small, enveloped virus containing single-stranded positive-sense RNA genome of approximately 15 kb [5, 18] . The viral genome contains 8 open reading frames (ORFs) that are transcribed in cells as a nested set of subgenomic mRNAs [5, 18, 19] . The ORF1a and ORF1b situated at the 5 H end of the genome represent nearly 75% of the viral genome and encode proteins with apparent RNA-dependent RNA polymerase activities [18, 20] .

In addition to the polymerase, the genomic sequence encodes 4 envelope glycoproteins named GP 2 (ORF2), GP 3 (ORF3), GP 4 (ORF4), and GP 5 (ORF5), as well as an 18±19 kDa unglycosylated membrane protein M (ORF6) and a 15 kDa nucleocapsid protein N (ORF7) [5, 15, 18, 19, 27] . The M protein contains 1±2 putative glycosylation sites and 3 highly hydrophobic regions in its N-terminal half [15, 17] , which are assumed to represent potential membrane spanning domains by analogy to the coronavirus M protein [23] . Another, 18 amino acid residues of the protein is probably exposed on the virion surface which relates to viral absorbtion and propagation [1] . The M protein is the most conserved structural protein between North American and European isolates. It is highly antigenic and can elicit a detectable antibody response as early as 10 days following infection [10, 14, 16, 32, 33] . Therefore it is suggested that the recombinant protein can be used as antigen for sera test and subunit vaccine. Because the structure and function of M protein are still unclear so far, it is necessary to prepare a large quantity of the recombinant M protein by the gene engineering method for studying the protein. Pichia pastoris has been developed into an expression system for high-level expression of recombinant proteins. It not only shares many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, but is also as easily manipulated as E. coli [7, 8, 11] . Many heterologous proteins have been highly expressed in P. pastoris such as TNF, EGF [4, 28] .

In the present study, we extracted the PRRSV total RNA, cloned the ORF6 gene by RT-PCR and sequenced the whole gene. Meanwhile, we inserted the native and modified ORF6 gene into the pPICZaA by the correct ORF and transformed the appropriate P. pastoris strain GS115. The modified gene was expressed on the higher level and more activity of secreted expression, but the native gene could not be expressed.

PRRSV CH-1a strain (obtained from Harbin Veterinary Research Institute, China) was propagated in the African green monkey kidney permissive cell line (MARC-145). Cells were cultured in Eagle's minimum essential medium (EMEM, Gibco, USA). Pichia expression vector pPICZaA and pichia strain GS115 came from invitrogen.

The PRRSV total RNA was isolated from MARC-145 cells infected with PRRSV CH-1a strain according to the manufacturer's recommendation (TaKaRa, Dalian). Based on the published complete nucleotide sequences of PRRSV VR2332 strain (GenBank accession No. UOO153), a pair of primers was designed for the cloning of ORF6 gene. Sequences of the primers were as follows: the sense primer was 5 H -GAAGGATCCGTTTCAGCGGAACAATGG-3 H and the antisense primer was 5 H -TGGAGCGGCCGCAG-CTGATTGACTGGCTGG-3 H . Reverse transcription (RT) was performed as follows: 10 ml total RNA, 1 ml corresponding antisense primer (10 mmol/L), 10 ml 5 Â RT-buffer, 1 ml RNase inhibitor (TaKaRa, Dalian), 16 ml 2.5 mmol/L dNTPs, 2 ml AMV Rtase (TaKaRa, Dalian) were mixed and incubated at 25 C for 10 min, 42 C for 1 h, and finally chilled on ice. To amplify the ORF6 gene, a mixture of the first stand cDNA, the corresponding sense and antisense primers, EX-Taq DNA polymerase, 10 Â PCR buffer, dNTPs, and sterile distilled water were added to a total volume of 50 ml. PCR was performed in Thermocycler with the following conditions: 94 C 4 min; 94 C 1 min, 56 C 1 min, 72 C 1.5 min, 35 cycles; extension at 72 C for 10 min. The purified specific fragment was cloned into pMD18-T vector and identified by PCR and specific restriction enzyme digestion. The positive recombinant plasmids were sequenced.

Based on the P. pastoris codon bias, poly (A) signal of yeast expression system and secondary structure of 5 H -end of foreign gene [12, 25, 26] , the ORF6 gene was synthesized, EcoRI and NotI sites were added to both ends of the modified ORF6 gene. The modified ORF6 gene and pPICZaA were digested with the restriction enzyme EcoRI and NotI, respectively. The interesting fragments were recovered and ligated. In the meantime, the native ORF6 gene was also inserted into the pPICZaA vector within correct ORF by the same methods.

Yeast vector pPICZaA/pPICZaA-ORF6/pPICZaA-M were digested with SacI enzyme and transformed competent pichia cells GS115 according to Easyselect pichia Expression kit instruction manual. Positive recombinant yeasts were selected on the Yeast Extract Peptone Dextrose Medium with Sorbitol (YPDS) plates with 100 mg Zeocin and the mut phenotype was confirmed on the selection Minimal Methanol Medium and Histidine (MMH) plates and Minimal Dextrose Medium and Histidine (MDH) plates. At the same time, colonies were picked into Yeast Extract Peptone Dextrose (YPD) medium and incubated overnight. The yeasts were lysed with Zymolyase (Sigma, USA) and gently extracted with an equal volume of pheno: chloroform (1 : 1, v/v) followed by an equal volume of chloroform: isoamyl alcohol (24 : 1, v/v). The aqueous layer was split into microcentrifuge tubes and added 1/2 volume of ammonium acetate and 2 volumes of ethanol to each tube, centrifuged at 10,000g for 20 min at 4 C. The pellets were washed once with 1 ml of 75% ethanol. After brief air-drying, the pellets were resuspended with 50 ml of TE buffer (5 mmol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA) each tube for PCR amplification. PCR conditions were: 95 C 4 min; 94 C 1 min, 55 C 1 min, 72 C 1.5 min, 35 cycles; extension at 72 C for 10 min.

A single colony was incubated in 25 ml of Buffered Glycerol-complex Medium (BMGY) in a 250 ml baffled flask, grown at 28±30 C in a shaking incubator for approximately 16±18 h until OD 600 of the culture reached 2±6. The cells were harvested and the cell pellets were resuspended to an OD 600 of 1.0 in Buffered Methanol-complex Medium (BMMY) and cultured at 28±30 C for 4 days. Methanol (100%) was added to a final concentration of 0.5% every 24 h. 1 ml of the expression culture was moved to a 1.5 ml micro centrifuge tube, centrifuged at maximum speed for 2±3 min at room temperature and the supernatant was transferred to a separate tube and analyzed for protein expression by Coomassie Blue staining SDS-PAGE and Western blot.

SDS-PAGE and Western blot were carried out by the procedure of Sambrook [24] Five Microliter of the supernatant was lysed in 5.0 ml of 2 Â SDS-PAGE loading buffer (0.2% bromophenol, 20% glycerol, 4% SDS, 100 mmol/L Tris-HCl, pH 6.8, 200 mmol/L dithiothreitol) by boiling for 10 min. Then samples were separated in the 15% polyacrylamide gel. Mock-yeast supernatant was similarly processed. Following SDS-PAGE, the gels were stained by Coomassie Blue or the proteins were transferred to nitrocellulose, using a semidry apparatus (Bio-Rad, Hercules, CA). The nitrocellulose blots were saturated with PBS containing 3% (W/V) BSA and 0.05% Tween20 overnight at 4 C. Recombinant proteins were incubated with porcine anti-PRRSV serum (1 : 50 dilution) in blocking solution for 1 h at 37 C, and followed by washing three times of 15 min each with PBS containing 0.05% Tween20. The blots were then incubated with alkaline phosphatase conjugated rabbit anti-swine IgG for 1 h at 37 C. After washing, the blots were developed using 5 Hbromo-4-chloro-3-indolyphosphate p-Toluidine salt (BCIP) and nitroblue tetrazolium chloride (NBT) (Gibco, USA).

Samples collected at different time points were analyzed for the concentration of both the total protein and interesting proteins to determine the optimal harvesting time. The total protein of each sample was measured, and the concentration of the interesting protein was calculated by scanning the Coomassie-stained SDS-PAGE and determined the optimal time post-induction to harvest. Based on the optimal harvesting time post-induction, the recombinant pichia strains were induced by methanol with a final concentration of 0.125%, 0.25%, 0.5%, 1.0%, respectively, to determine the optimal methanol concentration for inducible expression.

Nucleotide sequencing analysis indicated that the inserted fragment contained the entire coding sequence of PRRSV ORF6 gene. Furthermore, yeast has an optimal codon bias for the high-level expression. Yeast bias codons were scattered in the ORF6 gene. Since further site-directed mutagenesis was not feasible, we decided to chemically synthesize the entire ORF6 gene. In the design of the synthetic ORF6 gene, three aspects were considered. First, G C content was increased and special attention was paid to eliminate A T-rich runs for avoiding transcript stop. Second, the redundancy of the genetic code was used to insert unique KpnI site and identify modified or native gene. Third, the synthetic gene was designed to have an optimal codon bias for highlevel expression in yeast. No amino acid changes were made compared to the native sequence. In addition, EcoRI and NotI sites were added to both ends of the gene, respectively. Native and modified ORF6 genes were shown in Fig. 1 .

Modified ORF6 gene was inserted into the yeast expression vector pPICZaA by the unique EcoRI and NotI sites with the correct ORF to construct the yeast transfer vector. Native gene was subcloned into the pPICZaA vector by the same methods Fig. 2 

Since Zeocin gene exists in recombinant yeast but not in the parent strain GS115, 10 transforms were picked up to the YPD culture with 25 mg/ml Zeocin and grown at 28±30 C overnight. The total DNA of yeast was extracted and the interesting fragments were amplified by 2 pairs of primers: 5 H AOX1/3 H AOX1 and a-Factor/3 H AOX1. Results showed that native and modified ORF6 gene had been correctly integrated into the yeast genome.

A distinct polypeptide with a molecular mass (Mr) of approximately 23 kDa was detected in the recombinant supernatant and no other polypeptides were secreted (Fig. 3A) ; however the native ORF6 gene could not be expressed (data not shown). Recombinant M protein had immunogenic activity by analysis of Western blot (Fig. 3B) .

Five Microliter of the supernatant at different time points were analyzed by SDS-PAGE and the optimal induction time was found to be approximately 84 h. Protein band of lane 4 was about 84% of the total protein and the level of expression was approximately 2.0 g/L (Fig. 4) . Based on the optimal inducible time, the recombinant yeast was induced by methanol with a final concentration of 0.125%, 0.25%, 0.5%, 1.0%, respectively, and the results showed that the optimal methanol concentration was about 0.5%. The recombinant yeast could almost not be expressed with the final concentration of 0.125% and 0.25%. The expression levels were the same with 1% methanol concentration and 0.5% methanol concentration (Fig. 5 ).

It is necessary to express M protein in vitro in order to research its function and develop possibility as subunit vaccine. At present, GP3, GP5, and N proteins were expressed, but M protein could not be expressed in the baculovirus system [22] or its expression level was very low because the M protein was a highly hydrophobic protein [13] . As a eukaryote, P. pastoris has been developed as an excellent host for the production of foreign proteins [8] . The organism has the potential for high level of expression, efficient secretion, easily grown to very high cell densities and the advantages of higher eukaryotic expression systems such as protein processing, protein folding, posttranslational modification while being as easily as to be manipulated as E. coli or Saccharomyces cerevisiae [4, 7, 8, 11, 28] . It is faster, easier, and less expensive to be used than other eukaryotic expression systems such as baculovirus or mammalian tissue culture. As a yeast expression system, it uses the promoter from the methanol-induced alcohol oxidase geneÐAoxl, Fig. 2 . Schematic representation of the cloning strategy. Total RNA from PRRSV CH-1a strain-infected cells was used in the RT-PCR reaction to amplify ORF6 gene and the ORF6 gene was modified (see Materials and Methods). a-factor sequence was located between 5 H AOX1 and multiple cloning site (MCS). 5 H AOX1 A 942 bp fragment containing the AOX1 promoter that allows methanol-inducible, high-level expression in pichia; AOXTT AOX1 transcription termination. It is native transcription termination and polyadenylation signal from AOX1 gene ($260 bp) that permits efficient 3 H mRNA processing including polyadenylation for increased mRNA stability. shares the advantages of molecular and genetic manipulation with saccharomyces and has the added advantage of 10±100-fold higher heterologous protein expression level. These features make pichia very useful as a protein expression system [2, 11, 31] . In this paper, we have highly expressed the M protein with the yeast expression system, while native ORF6 gene was not expressed (data not shown).

We found that there were many factors drastically influencing protein production in this system including the copy number of the expression cassette, site, and model of chromosomal integration of expression cassette, mRNA 5 H -and 3 H -untranslated regions, translational start codon (AUG) context, A T composition of cDNA, transcriptional and translational blocks, nature of secretion signal, endogenous protease activity, host strain physiology, medium and growth conditions, and fermentation parameters, etc. [29] . In addition, an apparent difference of bias between yeast and bacteria was found by analyzing the codon characteristics of 110 yeast's genes. The usage frequency of codon AGA and CGC were 86.6% and 1.25%, respectively, and CGA and CGG were not used within the six codons of arginine [10, 25, 26] . So codons of arginine in the ORF6 gene were changed into AGA. Because codons of other amino acids were similar to arginine, we decided to modify the whole ORF6 gene. All codons of ORF6 gene were mutated according to yeast bias codons. In the meanwhile, rich ATT region was removed to avoid the undesirable termination signal of yeast. Results confirmed that the modified ORF6 gene was highly expressed (approximately 2.0 g/ L), indicating that ORF6 gene was successfully modified.

In this study, yeast-expressed protein M ( Fig. 3A  and B ) was about 23 kDa which is 4 kDa larger than the calculated molecular mass of 19 kDa. It is possibly glycoslated in yeast system because ORF6 gene of CH-1a strain has two latent glycoslated sites that are un-functional [30] . So the recombinant M protein was cut with Endoglycosylase H (Roche, USA), but M protein mass was not reduced (data not shown), Drew et al. (1995) also identified a nonglycosylated M protein in the transcription and translation studies of ORF6 gene [9] . Our studies further confirmed that the M protein expressed in the yeast system was a non-glycosylated protein.

Recombinant baculovirus-expressed M protein [13] was also larger than M detected in cell lysates [21] , in purified LV particles [19] or by in vitro translation of the Canadian and European strain of PRRSV [15, 19] . But in vitro translation of the LV M protein from infected cell lysate yielded a peptide of Mr 21.5 kDa [19] that was similar to the yeast-expressed M protein reported herein ( Fig. 3A  and B) .

In conclusion, a secreted yeast engineering strain was constructed, and the optimal induction conditions were determined for M protein of PRRSV. It is very convenient to separate and purify the M protein and product a large amount of the recombinant M protein by the fermentation and it provides a tool for studying its structural and functional characterization. 

Proceedings of the International Symposium on PRRS and Aujeszky's Disease

Molecular Cloning: A Laboratory Manual

This research was supported by the grants for the 973 National Program (strategic grant G1999011902). We thank Dr. Guo Aizhen for critical modification of the manuscript.