Elliott S, Fagin K D, Narhi L O, Miller J A, Jones M, Koski R, Peters M, Hsieh P, Sachdev R, Rosenfeld R D
Amgen Inc., Thousand Oaks, California 91320.
J Protein Chem. 1990 Feb;9(1):95-104. doi: 10.1007/BF01024990.
Recombinant human insulin-like growth factor I (IGF-I) is efficiently expressed and secreted from Saccharomyces cerevisiae using a yeast alpha-factor leader to direct secretion. However, approximately 10-20% of the IGF-I was in a monomeric form, the remaining materials being disulfide-linked aggregates. When the purified material was subjected to reverse-phase high-performance liquid chromatography (rp-HPLC), it gave two doublet peaks, I and II. Upon reduction, doublet peaks I and II converged to one doublet peak. This suggests that peaks I and II result from different disulfide structures, and the doublet feature of each peak results from other causes. Different disulfide structures between peaks I and II were also suggested from the near UV circular dichroism of these proteins. Only the peak II was biologically active, indicating that peak II has the correct disulfide structure. Concanavalin A affinity chromatography of the purified peak II doublet showed binding of the subpeak with an earlier rp-HPLC retention time, indicating that it was glycosylated. Sequence analysis of tryptic peptides suggested that Thr29 was the site of glycosylation. Site-directed mutagenesis was used to convert Thr29 to Asn29. This substitution reduced, but did not eliminate IGF-I glycosylation, suggesting additional glycosylation sites. The site of carbohydrate addition was consistent with the model that O-glycosylations occur on hydroxyl amino acids near proline residues in beta-turns.
重组人胰岛素样生长因子I（IGF-I）利用酵母α-因子前导序列指导分泌，可在酿酒酵母中高效表达和分泌。然而，约10%-20%的IGF-I呈单体形式，其余物质为二硫键连接的聚集体。当对纯化后的物质进行反相高效液相色谱（rp-HPLC）分析时，出现了两个双峰，即峰I和峰II。还原后，峰I和峰II合并为一个双峰。这表明峰I和峰II是由不同的二硫键结构导致的，而每个峰的双峰特征是由其他原因造成的。这些蛋白质的近紫外圆二色性也表明峰I和峰II之间存在不同的二硫键结构。只有峰II具有生物活性，这表明峰II具有正确的二硫键结构。对纯化后的峰II双峰进行伴刀豆球蛋白A亲和色谱分析，结果显示保留时间较早的亚峰具有结合能力，表明它被糖基化了。胰蛋白酶肽段的序列分析表明，苏氨酸29是糖基化位点。采用定点诱变将苏氨酸29转换为天冬酰胺29。这种替换减少了，但并未消除IGF-I的糖基化，这表明还存在其他糖基化位点。碳水化合物添加位点与β-转角中脯氨酸残基附近的羟基氨基酸上发生O-糖基化的模型一致。
