Development of molecularly imprinted polymer-assisted refolding and stabilization system

对于使用异源表达系统生产活性蛋白，包涵体中蛋白质的再折叠往往由于其产量低而成为瓶颈。在这项研究中，我们发现分子印迹聚合物（MIP）对天然溶菌酶促进了化学变性溶菌酶的折叠。以1M丙烯酰胺、1M甲基丙烯酸、1M 2-（二甲胺）甲基丙烯酸乙酯和5mg /ml溶菌酶为原料制备的MIP，能有效促进溶菌酶的再折叠，而非印迹聚合物则不能。将15 mg的MIP加入0.3 mg未折叠的溶菌酶中，可达到90%的再折叠率。我们还制备了胃蛋白酶的MIP，因为胃蛋白酶没有该序列就不能在成熟状态下进行折叠。我们尝试用MIP将变性胃蛋白酶重新折叠成成熟的形式。在MIP存在下，胃蛋白酶的CD谱与天然胃蛋白酶的CD谱几乎相同，表明其三级结构在恢复过程中几乎被恢复。然而，再生胃蛋白酶的催化活性仅为天然胃蛋白酶的15%，这表明只有一小部分再生胃蛋白酶恢复了其活性形式。预计MIP将识别蛋白质的天然形式，但其变性形式。利用热稳定蛋白的这一特性，构建了热稳定蛋白的筛选系统。制备的大肠杆菌核糖核酸酶HI (RNase HI) MIP用于此目的。将野生型RNase HI和热稳定突变型RNase HI （5H-RNase HI）与MIP一起在不同温度下孵育。电泳分析表明，在温度高于60℃时，MIP仅与5H-RNase HI结合。因此，该MIP将有助于选择热稳定突变蛋白。

For production of active proteins using heterologous expression systems, refolding of proteins from inclusion bodies often creates a bottleneck due to its poor yield. In this study, we show that molecularly imprinted polymer (MIP) toward native lysozyme promotes the folding of chemically denatured lysozyme. The MIP, which was prepared with 1M acrylamide, 1M methacrylic acid, 1M 2-(dimethylamino)ethyl methacrylate, and 5 mg/ml lysozyme, successfully promoted the refolding of lysozyme, whereas the non-imprinted polymer did not. The refolding yield of 90% was achieved when 15 mg of the MIP was added to 0.3 mg of the unfolded lysozyme.We also prepared MIP for pepsin, which cannot refold in its mature form without the prosequence. We tried to refold the denatured pepsin in its mature form using the MIP. The CD spectrum of pepsin renatured in the presence of the MIP was almost identical to that of the native pepsin, suggesting that its tertiary structure is almost recovered during the renaturation process. However, the catalytic activity of the renatured pepsin was only 15 % of that of the native pepsin, suggesting that only a small population of the renatured pepsin restores its active form.MIP is expected to recognize a protein in its native form but its denatured form. Taking advantage of such a property, we tried to construct a selection system for thermostabilized protein. The MIP prepared toward E. coli ribonuclease HI (RNase HI) was used for this purpose. The wild-type RNase HI and thermostabilized mutant RNase HI (5H-RNase HI) was incubated together with the MIP at various temperatures. Electrophoresis analysis showed that the MIP binds only 5H-RNase HI at temperatures higher that 60 ℃. Therefore, this MIP will be useful to select thermostabilized mutant proteins.