Modification of SARS-CoV S1 gene render expression in Pichia pastoris.
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.
Lu
Haisong
H
Provincial Key Laboratory of Preventive Veterinary Medicine, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China.
Yang
Guoliang
G
Fei
Xiaozhan
X
Guo
Hongyan
H
Tan
Yadi
Y
Chen
Huanchun
H
Guo
Aizhen
A
D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D008780:Methyltransferases / Q000235:genetics*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D020365:RNA Helicases; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*; D014170:Transformation, Genetic; D017361:Viral Nonstructural Proteins
D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D008780:Methyltransferases; C504965:Nsp13 protein, SARS-CoV; D020365:RNA Helicases