Effects of Fibronectin-Type V Collagen Recombinant Fusion Protein on Cell Adhesion and Cell Proliferation

Abstract

An expression vector pTF7520-Col-V-In, which encodes a fusion protein of the cell-binding domain of fibronectin (C277) and the insulin- and heparin-binding domain of the α1 chain of human type V collagen, was constructed. E. coli transfected with this plasmid synthesized a 50-kDa fusion protein. This fusion protein, C277-V, was purified from the crude extract by a single step heparin HPLC.Similar amounts of insulin bound to purified C277-V and to theα1 chain of type V collagen as judged by the binding of peroxidase-conjugated insulin. Cell-adhesive activity of C277-V was lower than that of the original fibronectin fragment C274, but similar numbers of cells adhered to both protein substrates when the culture dishes were coated with 1 mM of each protein. Insulin bound to the C277-V substratum stimulated the growth of mouse mammary tumor MTD cells in serum-free culture medium.