Zhang Y, Olsen D R, Nguyen K B, Olson P S, Rhodes E T, Mascarenhas D
Department of Molecular and Cell Biology, Celtrix Pharmaceuticals, Inc., Santa Clara, California 95054, USA.
Protein Expr Purif. 1998 Mar;12(2):159-65. doi: 10.1006/prep.1997.0834.
At the optimum temperature for its growth (37 degrees C), Escherichia coli tends to accumulate heterologous proteins in insoluble form. Fusion protein technology has been used to increase the solubility of overexpressed proteins in this organism, but with variable degrees of success. Fusion to a mutant form of DsbA (DsbAmut) confers higher levels of solubility to heterologous proteins in a reproducible way, even when E. coli is grown at 37 degrees C. We have shown this to be true with a diverse sample of eukaryotic proteins: IGF-I, IGFBP-3, 3C proteinase, TGF beta-2, sTGF beta-RII, BDNF, GDNF, mEGFBP, leptin, and GFP. In addition, we have investigated the effects of charge average and proline content on the solubility of DsbAmut fusions. Coexpression of a protein prolyl isomerase [cyclophilin (L-)] and modification of selected asparagine residues to aspartic acid appear to have beneficial effects on the accumulation of soluble heterologous proteins.
在其生长的最佳温度（37摄氏度）下，大肠杆菌倾向于以不溶性形式积累异源蛋白。融合蛋白技术已被用于提高该生物体中过表达蛋白的溶解度，但成功程度各不相同。与突变形式的DsbA（DsbAmut）融合以可重复的方式赋予异源蛋白更高水平的溶解度，即使大肠杆菌在37摄氏度下生长也是如此。我们已通过多种真核蛋白样本证明了这一点：胰岛素样生长因子-I（IGF-I）、胰岛素样生长因子结合蛋白-3（IGFBP-3）、3C蛋白酶、转化生长因子β-2（TGF beta-2）、可溶性转化生长因子β受体II（sTGF beta-RII）、脑源性神经营养因子（BDNF）、胶质细胞源性神经营养因子（GDNF）、小鼠表皮生长因子结合蛋白（mEGFBP）、瘦素和绿色荧光蛋白（GFP）。此外，我们还研究了电荷平均值和脯氨酸含量对DsbAmut融合蛋白溶解度的影响。共表达一种蛋白脯氨酰异构酶[亲环素（L-）]以及将选定的天冬酰胺残基修饰成天冬氨酸似乎对可溶性异源蛋白的积累具有有益影响。
