Seno M, Sasada R, Iwane M, Sudo K, Kurokawa T, Ito K, Igarashi K
Biotechnology Laboratories, Takeda Chemical Industries, Ltd., Osaka, Japan.
Biochem Biophys Res Commun. 1988 Mar 15;151(2):701-8. doi: 10.1016/s0006-291x(88)80337-1.
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.
利用定点诱变技术，将人碱性成纤维细胞生长因子（bFGF）成熟蛋白中位于氨基酸残基26、70、88和93处的四个半胱氨酸分别逐个替换为丝氨酸。当用丝氨酸取代bFGF蛋白70或88位的半胱氨酸残基时，其生物学活性和肝素结合能力得以保留。这一发现表明这些位置的半胱氨酸对于表达生物学活性并非必不可少。这些位置残基的替换，尤其是88位的替换，即使在用过氧化氢氧化后，也减少了从肝素亲和柱洗脱的bFGF多个峰所识别的异质性，这表明这些位置的半胱氨酸暴露于分子表面以形成诱导异源构象的二硫键。此外，在酸性条件下，这些修饰的bFGF在维持其活性方面表现得更加稳定。这些事实表明该蛋白已通过蛋白质工程成功修饰。
