Publication in refereed journal
香港中文大学研究人员 ( 现职)
| 韦妙宜教授 (那打素护理学院) |
| 徐国荣教授 (生物医学学院) |
| 冯国培教授 (生物医学学院) |
| 黄智?博士 (肿瘤学系) |
全文
| 数位物件识别号 (DOI) http://dx.doi.org/10.1007/s11262-008-0292-3 |
引用次数
Web of Sciencehttp://aims.cuhk.edu.hk/converis/portal/Publication/2WOS source URL
其它资讯
摘要To establish a rapid and economical method for the expression of viral proteins in high yield and purity by Pichia pastoris, the S protein of the SARS-CoV was selected in this study. Six S glycoprotein fragments were expressed in Escherichia coli BLhttp://aims.cuhk.edu.hk/converis/portal/Publication/21 and yeast KM71H strains. After purification by affinity chromatography, the protein identities were confirmed by western blot analysis, N-terminal sequencing and mass spectrometry. The proteins expressed in E. coli were low in solubility and bound by GroEL. They still formed soluble aggregates even when the GroEL was removed by urea. The proteins expressed in P. pastoris were relatively soluble. The maximal yield of the RBD reached 46 mg/l with purity greater than 95%. Pull-down assay revealed that ACEhttp://aims.cuhk.edu.hk/converis/portal/Publication/2 was specifically captured from cell lysate, indicating that the RBD was biologically active. The glycosylated and deglycosylated RBD was then subjected to SEC and results showed that deglycosylated RBD formed soluble aggregates again. Taken together, pure and biological active RBD of the S protein could be expressed in P. pastoris, and the P. pastoris expression platform will be a good alternative for the expression of viral proteins, in particular, the highly glycosylated surface proteins that mediate the tissue tropism and viral entry.
着者Chuck CP, Wong CH, Chow LMC, Fung KP, Waye MMY, Tsui SKW
期刊名称Virus Genes
出版年份http://aims.cuhk.edu.hk/converis/portal/Publication/2009
月份http://aims.cuhk.edu.hk/converis/portal/Publication/2
日期1
卷号38
期次1
出版社Springer Verlag (Germany)
页次1 - 9
国际标準期刊号09http://aims.cuhk.edu.hk/converis/portal/Publication/20-8569
语言英式英语
关键词ACEhttp://aims.cuhk.edu.hk/converis/portal/Publication/2; Pichia pastoris; RBD; SARS-CoV; Spike
Web of Science 学科类别Genetics & Heredity; GENETICS & HEREDITY; Virology; VIROLOGY
