A2 domain of human von Willebrand factor expressed in E. coli and its biological activity.
- Author:
Jian SU
1
;
Xia BAI
;
Wei-Qiang GAO
;
Chang-Geng RUAN
Author Information
1. Jiangsu Provincial Institute of Hematology, The First Affiliated Hospital of Suzhou University, Suzhou 215006, China.
- Publication Type:Journal Article
- MeSH:
ADAM Proteins;
metabolism;
ADAMTS13 Protein;
Escherichia coli;
genetics;
Humans;
Peptide Fragments;
biosynthesis;
genetics;
Purpura, Thrombotic Thrombocytopenic;
diagnosis;
metabolism;
Recombinant Proteins;
biosynthesis;
isolation & purification;
von Willebrand Factor;
biosynthesis;
chemistry;
genetics
- From:
Journal of Experimental Hematology
2005;13(6):1090-1093
- CountryChina
- Language:Chinese
-
Abstract:
Von Willebrand factor (vWF) is the unique substrate for the metalloprotease, ADAMTS-13, and plays a pivotal role in the pathology of von Willebrand disease (vWD) and thrombotic thrombocytopenic purpure (TTP). To study the pathogenesis of TTP and to establish a method to diagnose TTP, the DNA fragment of vWF-A2 domain was amplified and inserted into expression vector with 6 x His tag (pQE-30), the recombinant expression vector was transformed into E. coli (strain M15) and induced by IPTG. The recombinant fragment comprising residues 718-905 of mature vWF was designated as rvWF-A2. It was purified by Ni-NTA resin column chromatography and refolded in Tris buffer containing GSH and GSSG. The results demonstrated that rvWF-A2 was expressed successfully in E. coli M15, amounting to 42% of total bacterial protein with the purity over 98%. It was identified that rvWF-A2 can be efficiently cleaved by the citrated normal plasma while no cleavage can be detected by the TTP plasma or plasma with EDTA. It is concluded that rvWF-A2 expressed efficiently in E. coli demonstrated excellent biological activity, which lays a solid foundation for establishment of method to measure quantatively the activity of ADAMTS-13.
