Production in Escherichia coli of a biologically active subfragment of von Willebrand factor corresponding to the platelet glycoprotein Ib, collagen and heparin binding domains.
A full-length cDNA for vWF has been cloned from a human lung cDNA library and a fragment of this cDNA has been modified to allow its expression in E. coli. This fragment, which corresponds to Val 449-Asn 730 of vWF and includes the GPIb-binding domain and binding sites for collagen and heparin, was subcloned into an expression vector containing an inducible lambda PL promoter. On induction, the expressed recombinant vWF subfragment migrated with a mol wt of around 38,000 after SDS-PAGE. It was identified as a vWF fragment by Western blotting using either a polyclonal or a monoclonal antibody which inhibits the binding of vWF to GPIb. Following solubilization in urea, the bacterial extract inhibited ristocetin-induced platelet aggregation and bound to ristocetin-treated platelets, to collagen and to heparin.
Piétu
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INSERM U. 143, Hôpital de Bicêtre, le Kremlin Bicêtre, France.
Meulien
P
P
Cherel
G
G
Diaz
J
J
Baruch
D
D
Courtney
M
M
Meyer
D
D
D001665:Binding Sites; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D005796:Genes; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D010446:Peptide Fragments / Q000378:metabolism; D010957:Plasmids; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D015183:Restriction Mapping; D014841:von Willebrand Factor / Q000235:genetics* / Q000378:metabolism
D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; D006493:Heparin; D003094:Collagen