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Expression of a recombinant A1 domain of human VWF in COS 7 cells for conformational studies
Author(s) -
Chudapongse Nuannoi,
Leelayuwat Chanvit,
Kermode John C.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb104-d
Subject(s) - recombinant dna , domain (mathematical analysis) , chemistry , microbiology and biotechnology , biochemistry , biology , gene , mathematics , mathematical analysis
Von Willebrand factor (VWF) is a multimeric, plasma glycoprotein that plays an important role in hemostasis and thrombosis. It promotes platelet adhesion to damaged vascular endothelium. The A1 domain in VWF contains multiple binding sites, including those for platelet glycoprotein Ib, heparin, and the artificial modulators ristocetin and botrocetin. The structure of this domain is critical to its function as several point mutations have been found within this domain in patients with type 2 von Willebrand disease (VWD). Conformational changes in the domain are a topic of intense interest. Differences in its structure as a result of natural mutations in VWD have been demonstrated by X‐ray crystallography of recombinant A1 domain fragments. Such studies, however, do not prove that the native A1 domain can undergo a change in conformation. We plan to examine the patterns of digestion of a recombinant A1 domain by a various proteases in the presence and absence of ristocetin to test this hypothesis. In the present study, we have cloned the A1 domain of human VWF from genomic DNA and have designed a construct with a cleavable hexahistidine tag to express the recombinant protein. This facilitates purification of the glycosylated protein after its expression in COS 7 cells. Cleavage of the tag will allow subsequent studies of conformational change to be performed with the protein in its native state.