重组人组织型纤溶酶原激活剂(rht-PA)及其突变体的纯化

稳定高效表达重组人组织型纤溶酶原激活剂 (rht PA)的CHO细胞株和表达组合突变体的细胞株进行了 3L转瓶培养 .将培养上清分别进行了Lys Sepharose 4B亲和层析和Zn2 + Sepharose 4B层析两步纯化 ,rht PA纯度提高了 5 34倍 ,比活达 2 5× 10 5IU mg ,产率为 73% ;突变体纯度提高了1119倍 ,比活达 5 9× 10 5IU mg ,产率为 6 9% .纯化产物SDS PAGE分析显示 ,rht PA和突变体基本都呈单一条带 ,扫描分析均达到 98%以上纯度 .rht PA和突变体在纯化系统中的行为作对照分析发现 ,突变体的构建思想在Lys Sepharose 4B亲和层析过程中有充分体现 .这两步层析组合是很好的纯化t PA及其突变体的方法 ,尤其是Lys Sepharose 4B纯化突变体效果更好

Purification of Recombinant Human Tissue-type Plasminogen Activator (rht-PA) and Its Mutant

The CHO rht PA engineering cell strain 4B 3 and its mutant F 48 were cultured in the 3 L roller bottles with 10% serum Dulbecco′s modified Eagle medium(DMEM). rht PA and its mutants were purified by Lys Sepharose 4B and zinc chelate Sepharose 4B affinity chromotography respectively, from the cell culture supernatants after 24 hours incubation. The rht PA product had a specific activity of about 2 5×10 5 IU/mg protein with an overall activity recovery of 73%. The specific activity of purified mutant was 5 9×10 5 IU/mg protein with an overall activity recovery of 69%. The purified products showed either a single peak or a single band by chromatography and SDS PAGE. The purity was over 98% measured by UV absorption method.