[Stable expression and characterization of the von Willebrand factor cleaving protease].
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.
Ma
Zhenni
Z
Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, Suzhou, China.
Dong
Ningzheng
N
Zhang
Jingyu
J
Su
Jian
J
Wang
Anyou
A
Ruan
Changgeng
C
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*
D011994:Recombinant Proteins; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human