title: Production and characterization of functional domains of human fibronectin expressed in Escherichia coli.
abstract: 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 CS1, was almost inactive with BHK. CH-296, which contained CS1 at the C-terminus of CH-271, was more active with B16-F10 than H-296 and C-CS1, 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.
authors.affiliation: Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Shiga.
authors.forename: F
authors.initials: F
authors.lastname: Kimizuka
authors.forename: Y
authors.initials: Y
authors.lastname: Taguchi
authors.forename: Y
authors.initials: Y
authors.lastname: Ohdate
authors.forename: Y
authors.initials: Y
authors.lastname: Kawase
authors.forename: T
authors.initials: T
authors.lastname: Shimojo
authors.forename: K
authors.initials: K
authors.lastname: Hashino
authors.forename: I
authors.initials: I
authors.lastname: Kato
authors.forename: K
authors.initials: K
authors.lastname: Sekiguchi
authors.forename: K
authors.initials: K
authors.lastname: Titani
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002455:Cell Division; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism*; D005353:Fibronectins / Q000096:biosynthesis*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping
chemical_list: D005353:Fibronectins; D011994:Recombinant Proteins