Expression in Escherichia coli of a recombinant fragment (Ile 914-Leu 1364) of human von Willebrand factor containing a collagen binding domain.
Previous studies have shown that a collagen binding domain of human von Willebrand factor (vWF) resides on a fragment named SpI obtained by digestion with Staphylococcus aureus V8 protease which corresponds to residues Gly 911-Glu 1365 of the mature plasma vWF subunit. We have subcloned a fragment of a full-length cDNA encoding vWF into an expression vector which uses an inducible lambda PL promoter. The predicted product expressed by this plasmid is a fusion protein consisting of 16 amino acids (aa) of the lambda cII protein and aa Ile 914-Leu 1364 of human vWF. This fusion protein was shown to be expressed as insoluble inclusion bodies by induced E. coli harbouring the recombinant vector and was partially purified from bacterial debris and renatured. Partially purified bacterial extract run on SDS-polyacrylamide gels contained a major band representing 50-70% of the visualized proteins and corresponded to the predicted fusion protein. This band reacted with both polyclonal antibodies against human vWF and monoclonal antibodies (MAbs) which recognize the SpI fragment of plasma vWF. The radiolabelled partially purified bacterial extract was shown to bind specifically to human fibrillar collagen types I and III. This binding, which was a function of radiolabelled ligand and collagen concentrations, did not occur on monomeric denatured collagen. It was inhibited by both unlabelled bacterial extract and plasma vWF and also by a MAb against vWF SpI, which has the property of inhibiting vWF/collagen interaction. Our data demonstrate that this recombinant vWF SpI fragment, which can be obtained in large amounts, is a useful model for localizing the epitopes of monoclonal antibodies and then for studying the mechanism of vWF/collagen interaction.
Jorieux
S
S
Laboratoire de Recherche sur l'Hémostase, CRTS Lille, France.
Piétu
G
G
Gaucher
C
C
Mischler
F
F
Meulien
P
P
Meyer
D
D
Mazurier
C
C
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014841:von Willebrand Factor / Q000096:biosynthesis* / Q000235:genetics
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D003094:Collagen; D004247:DNA