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

Content Provider	Springer Nature Link
Author	Lu, Haisong Yang, Guoliang Fei, Xiaozhan Guo, Hongyan Tan, Yadi Chen, Huanchun Guo, Aizhen
Copyright Year	2006
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.
Starting Page	329
Ending Page	335
Page Count	7
File Format	PDF
ISSN	09208569
Journal	Virus Genes
Volume Number	33
Issue Number	3
e-ISSN	1572994X
Language	English
Publisher	Kluwer Academic Publishers
Publisher Date	2006-01-01
Publisher Place	Boston
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	SARS-CoV S1 gene P. pastoris Expression Deglycosylation Hyperglycosylation Medical Microbiology Virology Plant Sciences
Content Type	Text
Resource Type	Article
Subject	Virology Genetics Medicine Molecular Biology

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