Stable expression and characterization of the von Willebrand factor cleaving protease.
- Author:
Zhenni MA
1
;
Ningzheng DONG
;
Jingyu ZHANG
;
Jian SU
;
Anyou WANG
;
Changgeng RUAN
Author Information
1. Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, Suzhou, China.
- Publication Type:Journal Article
- MeSH:
ADAM Proteins;
biosynthesis;
genetics;
metabolism;
ADAMTS13 Protein;
HeLa Cells;
Humans;
Recombinant Proteins;
biosynthesis;
genetics;
metabolism;
Transfection;
von Willebrand Factor;
metabolism
- From:
Chinese Journal of Biotechnology
2010;26(2):244-248
- CountryChina
- Language:Chinese
-
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.
