Arakawa T, Hsu Y R, Schiffer S G, Tsai L B, Curless C, Fox G M
Amgen Inc., Thousand Oaks, CA 91320.
Biochem Biophys Res Commun. 1989 May 30;161(1):335-41. doi: 10.1016/0006-291x(89)91601-x.
Using oligo site-directed mutagenesis, we have modified our synthetic gene for human basic fibroblast growth factor (bFGF) to replace all four cysteine codons with serine codons. The corresponding protein was expressed in Escherichia coli and purified from inclusion bodies by solubilization in urea followed by a series of column chromatographies and a folding step. The resulting protein, having no cysteine residues, is unable to form either intramolecular or intermolecular disulfide bonds. The secondary and tertiary structures of the purified analog, as determined by circular dichroism and fluorescence spectroscopy, were identical within experimental error to recombinant bovine and human bFGF with unaltered amino acid sequences. Reflecting the similar conformation, the analog protein exhibited mitogenic activity on NIH 3T3 cells which was indistinguishable from the natural sequence molecule.
利用寡核苷酸定点诱变技术，我们对人碱性成纤维细胞生长因子（bFGF）的合成基因进行了修饰，将所有四个半胱氨酸密码子替换为丝氨酸密码子。相应的蛋白质在大肠杆菌中表达，并通过在尿素中溶解从包涵体中纯化，随后进行一系列柱层析和折叠步骤。所得蛋白质没有半胱氨酸残基，无法形成分子内或分子间二硫键。通过圆二色性和荧光光谱测定，纯化类似物的二级和三级结构在实验误差范围内与氨基酸序列未改变的重组牛和人bFGF相同。反映出相似的构象，该类似物蛋白在NIH 3T3细胞上表现出与天然序列分子无法区分的促有丝分裂活性。
