Development of molecularly imprinted polymer-assisted refolding and stabilization system

2006 Fiscal Year Final Research Report Summary

• Project/Area Number: 17560692
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biofunction/Bioprocess
• Research Institution: Nihon University
• Principal Investigator: HARUKI Mitsuru Nihon University, College of Engineering, Associate Professor, 工学部, 助教授 (30273593)
• Project Period (FY): 2005 – 2006
• Keywords: Molecularly imprinted polymer / Refolding / Lysozyme / Pepsin / Ribonuclease HI / Thermostability / 蛋白質耐熱化

Research Abstract

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.