Expression of the cell-binding domain of human fibronectin in E. coli. Identification of sequences promoting full to minimal adhesive function.
Two cDNA subfragments containing the cell-attachment site of human fibronectin (FN) were expressed as beta-galactosidase fusion proteins in E. coli. The products were purified to homogeneity by monoclonal antibody affinity chromatography and assayed for activity in a standard cell-adhesion assay. A fusion protein containing an 80 kDa fragment of human FN appeared functionally equivalent to intact FN purified from human plasma, whereas a truncated fusion protein of 33 kDa still containing a previously postulated cell-attachment site was approx. 50-fold less active. Our study establishes a system for analyzing adhesive protein function by DNA manipulation, rules out any major role for eukaryotic post-translational modifications in FN adhesive function, and localizes additional functional activity to a 1.3 kb region.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001681:Biological Assay; D002448:Cell Adhesion; D002460:Cell Line; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics*; D005353:Fibronectins / Q000235:genetics* / Q000502:physiology; D006801:Humans; D007668:Kidney; D010446:Peptide Fragments / Q000235:genetics / Q000502:physiology; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D001616:beta-Galactosidase
D005353:Fibronectins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase