A2 domain of human von Willebrand factor expressed in E. coli and its biological activity / 中国实验血液学杂志
Journal of Experimental Hematology
;
(6): 1090-1093, 2005.
Article
in Chinese
| WPRIM
| ID: wpr-343820
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Peptide Fragments
/
Purpura, Thrombotic Thrombocytopenic
/
Recombinant Proteins
/
Von Willebrand Factor
/
Chemistry
/
Diagnosis
/
Escherichia coli
/
ADAM Proteins
/
ADAMTS13 Protein
/
Genetics
Type of study:
Diagnostic study
/
Prognostic study
Limits:
Humans
Language:
Chinese
Journal:
Journal of Experimental Hematology
Year:
2005
Type:
Article
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