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

1991; Oxford University Press; Volume: 110; Issue: 2 Linguagem: Inglês

10.1093/oxfordjournals.jbchem.a123572

1756-2651

Fusao Kimizuku, Yuki Taguchi, Yoichi Ohdate, Yasutoshi Kawase, Tomoko Shimojo, Kimikazu Hashino, Ikunoshin Kato, Kiyotoshi Sekiguchi, Koiti Titani,

Protease and Inhibitor Mechanisms

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.