期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 59, 期 2, 页码 223-231出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2008.02.002
关键词
HIV-1; envelope; gp120; V3; protease; expression; protease inhibitors; CHO-K1 cells
Specific proteolytic cleavage of the gp120 subunit of the HIV-1 envelope (Env) glycoprotein in the third variable domain (V3) has previously been reported to occur in several cell lines, including Chinese hamster ovary cells that have been used for production of Env-based HIV vaccine candidates. Here we report that this proteolytic activity on JRCSF gp120 is dependent on cell density, medium conditions, and supernatant concentration. The resulting cleaved polypeptides cannot be separated from intact gp 120 by conventional or affinity chromatography under non-reducing conditions. Inhibitor studies reveal that Pefabloc and benzamidine, but not chymostatin, block gp120 cleavage in a dose-dependent fashion, suggesting the presence of a trypsin-like serine protease in CHO-K1 cells. The proteolytic activity is increased with certain types of cell culture growth media. A combination of serum-free OptiMEM media during expression and potent protease inhibitors post-expression can effectively prevent HIV gp 120 degradation. The same strategy can be applied to the expression and purification of gp 120 of other strains or other forms of envelope-based vaccine candidates containing V3 sequences. (C) 2008 Elsevier Inc. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据