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质，由于其产量低，从包涵体中复性蛋白质通常会产生瓶颈。在这项研究中，我们表明，分子印迹聚合物（MIP）对天然溶菌酶促进化学变性溶菌酶的折叠。用1 M丙烯酰胺、1 M甲基丙烯酸、1 M甲基丙烯酸二甲氨基乙酯和5 mg/ml溶菌酶制备的分子印迹聚合物成功地促进了溶菌酶的复性，而非印迹聚合物则没有。当0.3mg未折叠的溶菌酶中加入15 mg MIP时，复性率可达90%。我们尝试使用MIP将变性胃蛋白酶重折叠成其成熟形式。在MIP存在下复性的胃蛋白酶的CD光谱与天然胃蛋白酶的CD光谱几乎相同，表明其三级结构在复性过程中几乎被恢复。然而，复性的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复性的胃蛋白酶恢复其活性形式。预期MIP识别天然形式的蛋白质，但其变性形式。利用这一特性，我们试图构建一个热稳定蛋白的选择系统。制备的MIP具有良好的E.大肠杆菌核糖核酸酶HI（RNase HI）用于该目的。将野生型RNA酶HI和热稳定的突变体RNA酶HI（5 H-RNA酶HI）与MIP一起在各种温度下孵育。电泳分析表明，MIP在60 ℃以上仅与5 H-RNase HI结合。因此，该MIP将是有用的，以选择热稳定的突变蛋白。

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.