Cloning and expression of the metalloproteinase domain of human von Willebrand factor-cleaving protease
- VernacularTitle:血管性血友病因子裂解酶金属蛋白酶域的克隆和表达
- Author:
Weiqiang GAO
;
Xia BAI
;
Jianxin FU
;
Changgeng RUAN
- Publication Type:Journal Article
- Keywords:
von Willebrand factor;
Cloning, gene;
Gene expression
- From:
Chinese Journal of Pathophysiology
2000;0(10):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To clone and express the metalloproteinase domain of human von Willebrand factor-cleaving protease (vWF-cp). METHODS: The metalloproteinase domain of human vWF-cp, amplified from the plasmid containing the vWF-cp cDNA gene by using polymerase chain reaction, was cloned into pUC18, and its accuracy was verified by sequencing. Then the domain was inserted into the multiclone site of pET28a(+) and included a 6?His Tag at its amino terminal. After induced by IPTG, the recombinant protein was purified by using a Ni-NTA column and confirmed by Western blot. RESULTS: Comparison of the nucleotide sequence of our cloned domain with the GenBank sequence revealed no difference. High-level expression of the recombinant protein was yielded after 5-hour induction, which amounted to 28% of total bacteria protein in inclusion body. Western blot demonstrated that it possessed high specificity. CONCLUSION: The metalloproteinase domain of vWF-cp was high efficiently expressed in Escherichia coli. This might contribute to the further study of the relationship between its structure and function. [
