[A2 domain of human von Willebrand factor expressed in E. coli and its biological activity].
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.
Su
Jian
J
Jiangsu Provincial Institute of Hematology, The First Affiliated Hospital of Suzhou University, Suzhou 215006, China.
Bai
Xia
X
Gao
Wei-Qiang
WQ
Ruan
Chang-Geng
CG
D051722:ADAM Proteins / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D011697:Purpura, Thrombotic Thrombocytopenic / Q000175:diagnosis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014841:von Willebrand Factor / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D010446:Peptide Fragments; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human