Modification of SARS-CoV S1 gene render expression in Pichia pastoris

Abstract

S1 gene fragment containing receptor-binding region was amplified by several sets of primers using Over-Lap PCR. The native S1 gene was modified at A + T abundant regions; n.t.777-1683, n.t.1041-1050, n.t.1236-1248, n.t.1317-1335, n.t.1590-1605; based on the same amino acid sequences. The modified gene was cloned into a yeast expression vector pPIC9K. The resultant plasmid pPIC9K- S1 was transformed into Pichia pastoris GS 115 and the protein expression was induced with methanol. SDS-PAGE confirmed that the recombinant SI was secreted in the supernatant of induced GS 115. The protein yield reached 69 mg/l. ELISA and Western blot demonstrated that the S1 could react with the convalescent sera of people infected by SARS-CoV. Furthermore, ligand blot assay showed that the recombinant S1 could react with ACE2, the receptor of SARS-CoV. The molecular mass of expressed S1 was about 70 kDa, which was higher than that of the 30 kDa expected. PNGase F deglycosylation resulted in a protein band of 30 kDa. In conclusion, the S1 gene modification rendered the high-level expression of S1 in P. pastoris GS 115 and the protein was secreted as a biologically active form which was hyperglycosylated. © Springer Science+Business Media, LLC 2006.