Hsu Eric, Osslund Timothy, Nybo Rebecca, Chen Bao-Lu, Kenney William C, Morris C Fred, Arakawa Tsutomu, Narhi Linda O
Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
Protein Eng Des Sel. 2006 Apr;19(4):147-53. doi: 10.1093/protein/gzj013. Epub 2006 Feb 14.
Native sequence keratinocyte growth factor (KGF) is fairly unstable, as manifested by the loss of the monomeric native protein accompanied by the accumulation of aggregated species during storage at moderate temperatures. Several different types of analogs were generated and the storage stability of the protein assessed. In the first type of analog one or more of the five cysteinyl residues in KGF were replaced; in the second class the N-terminal residues that included the first disulfide bond were deleted. Both of these types of analogs involved removal of the disulfide bond between cysteines 1 and 15. The third group involved mutating one of the basic amino acids located in a cluster of positive charges (involved in heparin binding) around Arg144 to a neutral or acidic amino acyl residue. Among the cysteine replacement analogs, the double mutation of Cys1 and 15 to Ser resulted in significantly increased stability without compromising the mitogenic activity, while Cys to Ser mutations at other positions were either destabilizing or had no effect. Deletion of the 15, 23 or 27 N-terminal amino acyl residues also increased the stability of the protein. The activity of the analogs was not affected by the deletion of 15 or 23 amino acids, but it was significantly decreased upon removal of the 27 N-terminal amino acyl residues. Much greater stability was achieved by mutation of the basic amino acids, especially Arg144, to Glu or Gln, but this increase in stability was accompanied by large decrease in activity. The analog with the 23 N-terminal amino acyl residues deleted represents one of the best compromises between increased stability and retention of activity.
天然序列的角质形成细胞生长因子（KGF）相当不稳定，在中等温度下储存时，单体天然蛋白会丢失，同时聚集物会积累，这表明了其不稳定性。生成了几种不同类型的类似物，并评估了该蛋白的储存稳定性。在第一类类似物中，KGF中的五个半胱氨酸残基中的一个或多个被替换；在第二类中，包括第一个二硫键的N端残基被删除。这两类类似物都涉及去除半胱氨酸1和15之间的二硫键。第三组涉及将位于Arg144周围带正电荷簇（参与肝素结合）中的一个碱性氨基酸突变为中性或酸性氨基酸残基。在半胱氨酸替换类似物中，Cys1和15突变为Ser的双突变导致稳定性显著增加，同时不影响促有丝分裂活性，而其他位置的Cys突变为Ser要么使稳定性降低，要么没有影响。删除15、23或27个N端氨基酸残基也增加了该蛋白的稳定性。删除15或23个氨基酸对类似物的活性没有影响，但删除27个N端氨基酸残基后活性显著降低。将碱性氨基酸，尤其是Arg144突变为Glu或Gln可实现更高的稳定性，但这种稳定性的增加伴随着活性的大幅下降。删除23个N端氨基酸残基的类似物代表了稳定性增加和活性保留之间的最佳折衷之一。
