%0 Journal Article
%T Stable expression and characterization of the von Willebrand factor cleaving protease<br>血管性血友病因子裂解蛋白酶的稳定表达及其活性检测
%A Zhenni M
%A Ningzheng Dong
%A Jingyu Zhang
%A Jian Su
%A Anyou Wang
%A Changgeng Ruan
%A <br>马珍妮
%A 董宁征
%A 张敬宇
%A 苏健
%A 汪安友
%A 阮长耿
%J 生物工程学报
%D 2010
%I 
%X This study was to acquire recombinant protein of von Willebrand factor cleaving protease (ADAMTS13, a disintegrin and metalloprotease with a thromboSpondin type 1 motifs 13), for further studies on its biological function in thrombosis and hemostasis. We transfected the Hela cells with the plasmid pSecTag-ADAMTS13 by lipofectamine. A positive cell cloning was selected by hygromycin-B. The recombinant protein was purified with Ni-NTA agarose column by gradient imidazole. The purity and immune activity of purified products were identified with SDS-PAGE and Western blotting respectively. We also measured the enzymatic activity of recombinant protein (rADAMTS13) by GST-His two-site ELISA assay. The results showed that we successfully constructed Hela cells ADAMTS2-4 which expressed high level of rADAMTS13. We received about 5.8 mg recombinant protein in culture supernantants per liter purified with Ni-NTA column. The protein formed a main lane at the position of 190 kDa with SDS-PAGE and reacted with polyclonal antibody against ADAMTS13 by Western blotting. The amount of rADAMTS13 activity was 6.4 U/mL, according to the normal plasma defined as 1 U/mL. In conclusion, rADAMTS13 protein had high purity, immune activity and good enzymatic activity, which could establish the experimental foundation for further research on biological function and mechanism of this unique metalloprotease.
%K von Willebrand factor cleaving protease
%K eukaryotic expression
%K recombinant protein<br>血管性血友病因子裂解蛋白酶，真核表达，重组蛋白
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=A66E90C274451689E69F6F0291467824&aid=6A1382CBBBF0E16625EAA6D46D28A2B0&yid=140ECF96957D60B2&vid=96C778EE049EE47D&iid=0B39A22176CE99FB&sid=5B5B75F4854B8331&eid=FBA00558C57D9C11&journal_id=1000-3061&journal_name=生物工程学报&referenced_num=0&reference_num=0