High-yield expression of recombinant SARS coronavirus nucleocapsid protein in methylotrophic yeast Pichia pastoris.
OBJECTIVE
Nucleocapsid (N) protein plays an important role in reproduction and pathological reaction of severe acute respiratory syndrome (SARS) coronavirus (SCoV), the antigenicity of the protein is better than spike (S) protein. This study was to find a highly specific and antigenic recombinant SCoV nucleocapsid (rSCoVN) protein, and to provide a basis for further researches on early diagnosis of SARS.


METHODS
Full length cDNA of SCoV nucleocapsid (SCoVN) protein was amplified through polymerase chain reaction (PCR) and cloned into yeast expression vector pPIC3.5K to construct plasmid of pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into Pichia pastoris (P.pastoris) GS115 (His-Mut+) by electroporation. His(+)Mut(+) recombinant strains were identified by PCR and cultivated on MM/MD plates. The influence of different factors on biomass and rSCoVN protein production during induction phase, such as various induction media, dissolved oxygen (DO) and different final concentrations of methanol, was subsequently studied. The expression level and activation were detected by SDS-PAGE and Western-blot respectively.


RESULTS
All of the recombinants were His(+)Mut(+) after transformation of P.pastoris with linearized plasmids. The BMMY medium was optimal for recombinant ScoVN (rSCoVN) protein expression and growth of the recombinant strains. The final optimal concentration of methanol was 20 mL/L, the DO had a significant effect on rSCoVN protein expression and growth of recombinant strains. The rSCoVN protein expressed in recombinant strains was about 8% of the total cell protein, 520 mg/L of rSCoVN protein was achieved, and a maximum cell A at 600 nm of 62 was achieved in shake flask culture. The rSCoVN protein had a high specificity against mouse-anti-SARS-CoVN-mAb and SARS positive sera, but had no cross-reaction with normal human serum. The biological activity of rSCoVN expressed in P.pastoris was about 4-fold higher than that expressed in E.coli when the same rSCoVN protein quantity was used.


CONCLUSIONS
Active recombinant severe acute respiratory syndrome (SARS) coronavirus nucleocapsid (rSCoVN) protein can be successfully expressed in recombinant methylotrophic yeast P.pastoris GS115. The rSCoVN protein has a high specificity against SARS-CoVN-mAb and SARS positive sera, but has no cross-reaction with normal human serum. This provides a basis for further researches on the early diagnosis of SARS and the mechanism of SCoV.
Liu
Ru-Shi
RS
The Key Laboratory for Cell Biology and Tumor Cell Engineering of the Ministry of Education; The Research Center for Medical Molecular Virology of Fujian Province, Xiamen University, Xiamen 361005, Fujian Province, China.
Yang
Kun-Yu
KY
Lin
Jian
J
Lin
Yi-Wei
YW
Zhang
Zhi-Hong
ZH
Zhang
Jun
J
Xia
Ning-Shao
NS
D001431:Bacteriological Techniques; D003470:Culture Media / Q000378:metabolism / Q000494:pharmacology; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D000432:Methanol / Q000378:metabolism / Q000494:pharmacology; D019590:Nucleocapsid Proteins / Q000235:genetics*; D010641:Phenotype; D010843:Pichia / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*; D014170:Transformation, Genetic
D003470:Culture Media; D018076:DNA, Complementary; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins; D000432:Methanol