人组织型纤溶酶原激活剂(t-PA)突变体在CHO细胞中表达产物的纯化研究  

Purification of a combination mutant(FrGGI) of human tissue-type plasminogen activator(t-PA) from the culture medium of the expression cell

作　　者：赵庆国[1] 张郁 黄培堂[2] 刘士辉[2] 焦保权 

机构地区：[1]中国人民解放军白求恩国际和平医院,河北石家庄050082 [2]中国人民解放军军事医学科学院生物工程研究所,北京100071

出　　处：《解放军药学学报》2000年第3期117-119,共3页Pharmaceutical Journal of Chinese People's Liberation Army

摘　　要：目的 :为了从细胞株培养液中纯化长半衰期的t PA组合突变体FrGGI。方法 :采用高表达克隆细胞株 ,进行大量培养 ,收集 2 4h细胞培养液为初始材料 ,利用t PAK2区含有Lysine结合位点的特性和Zn2 +与蛋白质中咪唑、巯基等基团螯合作用 ,经Lysine Sepharose 4B亲和层析和Zn2 + Sepharose 4B金属螯合层析二步组合纯化。结果 :获得高比活的FrGGI,为研究该突变体生物学特性及推广应用奠定基础。结论 :利用赖氨酸和锌离子柱二步组合纯化方法 ,可有效地分离纯化tAim To purify a long half-life tPA combination mutant,which was deglycosylated from K1 and K2 domain,deleted PAI-1 binding site(residues Lys296 to Gly302) and designated as FrGGI.Methods The FrGGI was highlevel stable expressed in CHO cell through coamplification of the mutant and DHFR cDNA. The mutant protein was purified by Lysine sepharose 4B and Zn 2+ sepharose 4B affinity chromotography from the culture supernatant after 24 hours incubation.Result We have purified the FrGGI protein, it has high specific activity.This will make possible its biological analyses and application.Conclusion TPA and its mutants with K2 domain can be purified effectively using Lysine sepharose 4B and Zn 2+ sepharose 4B affinity chromotography.

关 键 词：组织型纤溶酶原激活剂 突变体 纯化 CHO细胞 

分 类 号：R973.2[医药卫生—药品]