title: [Stable expression and characterization of the von Willebrand factor cleaving protease].
abstract: 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.
authors.affiliation: Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, Suzhou, China.
authors.forename: Zhenni
authors.initials: Z
authors.lastname: Ma
authors.forename: Ningzheng
authors.initials: N
authors.lastname: Dong
authors.forename: Jingyu
authors.initials: J
authors.lastname: Zhang
authors.forename: Jian
authors.initials: J
authors.lastname: Su
authors.forename: Anyou
authors.initials: A
authors.lastname: Wang
authors.forename: Changgeng
authors.initials: C
authors.lastname: Ruan
mesh_terms: D051722:ADAM Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D000071120:ADAMTS13 Protein; D006367:HeLa Cells; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human