Production and Characterization of Functional Domains of Human Fibronectin Expressed in Escherichia coli

• Published in: Journal of biochemistry (Tokyo) Vol. 110; no. 2; pp. 284 - 291
• Main Authors: Kimizuku, Fusao, Taguchi, Yuki, Ohdate, Yoichi, Kawase, Yasutoshi, Shimojo, Tomoko, Hashino, Kimikazu, Kato, Ikunoshin, Sekiguchi, Kiyotoshi, Titani, Koiti
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.08.1991
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Biological and medical sciences; Blotting, Western; Cell Division; Cricetinae; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Escherichia coli - metabolism; Fibronectins - biosynthesis; Fundamental and applied biological sciences. Psychology; Glycoproteins; Humans; Molecular Sequence Data; Plasmids; Proteins; Recombinant Proteins - biosynthesis; Restriction Mapping; Human; Escherichia coli; Rodentia; Glycoproteins; Adhesion; Biological activity; Fibronectin; Vertebrata; Mammalia; Domain structure; Bacteria; Recombinant protein; Fibroblast; Hamster; Enterobacteriaceae
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a123572

An efficient expression system was constructed in Escherichia coli that produced a 33-kDa fragment, C-274, of human fibronectin with a strong cell-adhesive activity. The entire sequence of the heparin-binding domain with 271 amino acids, H-271, was also expressed. Deletion analysis of the type III repeats showed that the heparin-binding site was at type III-13. The cell-adhesive activity of a fusion protein, CH-271, containing the cell- and the heparin-binding domains was twice that of C-274 when BHK but not B16-F10 melanoma cells were tested; H-271 alone was inactive. Recombinant proteins containing the CS1 sequence of the IIICS region were more active than C-274 and CH-271 with B16-F10. However, H-296, which contained both H-271 and CSl, was almost inactive with BHK. CH-296, which contained CSl at the C-terminus of CH-271, was more active with B16-F10 than H-296 and C-CSl, which was produced by the deletion of H-271 from CH-296. Thus, the cell-binding domain was active with both kinds of cells. The heparin-binding domain promoted the adhesion of both kinds of cells only when linked to the cell-binding domain or CS1. CS1 was specific for the adhesion of B16-F10 but was not essential.