title: Stabilizing basic fibroblast growth factor using protein engineering.
abstract: Using site directed mutagenesis, each of the four cysteines present at amino acid residues 26, 70, 88, and 93 of the mature protein of human basic fibroblast growth factor (bFGF) was individually changed to serine. The biological activity and heparin binding ability was retained when the serine was substituted for the cysteine residue at either 70 or 88 of the bFGF protein. This finding indicates that the cysteines at these positions are not essential for expressing biological activity. The substitution of the residues at these positions, especially at position 88, reduced the heterogeneity recognized as several peaks of bFGF eluted from a heparin affinity column, even after oxidation with hydrogen peroxide, suggesting that the cysteines at these positions are exposed to the surface of the molecule to form disulfide bonds that induce heterologous conformations. Furthermore, under acidic conditions, these modified bFGFs are revealed to be more stable in maintaining their activity. These facts suggest that this protein has been successfully modified by protein engineering.
authors.affiliation: Biotechnology Laboratories, Takeda Chemical Industries, Ltd., Osaka, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Seno
authors.forename: R
authors.initials: R
authors.lastname: Sasada
authors.forename: M
authors.initials: M
authors.lastname: Iwane
authors.forename: K
authors.initials: K
authors.lastname: Sudo
authors.forename: T
authors.initials: T
authors.lastname: Kurokawa
authors.forename: K
authors.initials: K
authors.lastname: Ito
authors.forename: K
authors.initials: K
authors.lastname: Igarashi
mesh_terms: D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003545:Cysteine; D004262:DNA Restriction Enzymes; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D005346:Fibroblast Growth Factors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005818:Genetic Engineering; D006493:Heparin; D006801:Humans; D009154:Mutation; D010957:Plasmids; D012694:Serine
chemical_list: D012694:Serine; D005346:Fibroblast Growth Factors; D006493:Heparin; D004262:DNA Restriction Enzymes; D003545:Cysteine