Du Sean X, Xu Li, Viswanathan Sridhar, Whalen Robert G
Maxygen, Inc., Infectious Diseases, 515 Galveston Drive, Redwood City, CA 94063, USA.
Protein Expr Purif. 2008 Jun;59(2):223-31. doi: 10.1016/j.pep.2008.02.002. Epub 2008 Feb 21.
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 gp120 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 gp120 degradation. The same strategy can be applied to the expression and purification of gp120 of other strains or other forms of envelope-based vaccine candidates containing V3 sequences.
先前有报道称，在包括用于生产基于Env的HIV疫苗候选物的中国仓鼠卵巢细胞在内的几种细胞系中，HIV-1包膜（Env）糖蛋白的gp120亚基在第三个可变结构域（V3）中发生了特异性蛋白水解切割。在此我们报告，这种对JRCSF gp120的蛋白水解活性取决于细胞密度、培养基条件和上清液浓度。在非还原条件下，通过常规或亲和色谱法无法将产生的切割多肽与完整的gp120分离。抑制剂研究表明，苯甲磺酰氟和苯甲脒而非抑肽酶以剂量依赖性方式阻断gp120切割，这表明CHO-K1细胞中存在一种胰蛋白酶样丝氨酸蛋白酶。蛋白水解活性在某些类型的细胞培养生长培养基中会增加。在表达过程中使用无血清OptiMEM培养基并在表达后使用强效蛋白酶抑制剂的组合可以有效防止HIV gp120降解。相同的策略可应用于其他毒株的gp120或含有V3序列的其他形式的基于包膜的疫苗候选物的表达和纯化。
