Protein Refolding Assisted By Copolymerized And Grafted Poly (N-isopropylacrylamide) In Vitro

How to refold proteins expressed as inclusion bodies into correct conformation with high yield at high concentration is becoming one of the industrial bottlenecks in biological engineering field. In order to solve above problems, novel refolding technologies have been explored recently, and poly(N-isopropylacrylamide) (PNIPAM) hydrogel attracts much attention due to its thermo-sensitive characteristics. In this thesis, copolymerized and grafted PNIPAM were synthesized and then applied into refolding of lysozyme and recombinant human interferon-y (rhIFN-y) in vitro.Firstly, P(NIPAM-co-SA) gel particles were synthesized using inverse suspension polymerization by adding sodium acrylate (SA) to improve the phase transition properties of PNIPAM. The morphologies, size distribution and thermo-sensitive characteristics of gel particles were studied. When the protein concentration was 250μg/ml, the activity recovery of lysozyme increased to 76.5% assisted by P(NIPAM-co-SA) (A=4%) gel particles compared with 55.6% by simple dilution. When refolding finished, the gel particles could be removed and recovered easily and the activity recovery of lysozyme was still as high as 61.5% after reused for 5 batches. With the addition of different amounts of SA co-monomer, the hydrophobicity of the copolymer could be varied. Then the copolymerized hydrogel inhibits protein molecules aggregation more effectively through the moderate hydrophobic interactions between copolymers and protein intermediates in the course of lysozyme refolding compared with PNIPAM polymer.Secondly, PS-grafted-PNIPAM particles were synthesized by the non-soap emulsion polymerization and UV-initiation. The morphologies and thermo-sensitive characteristics of gel particles were studied. PS-grafted-PNIPAM particles were applied to assist lysozyme and rhIFN-y inclusion bodies refolding. The refolding conditions mediated by PS-grafted-PNIPAM hydrogel were optimized to increase the refolding yield. The results indicated that PS-grafted-PNIPAM particles could indeed assist lysozyme and rhIFN-y refolding and was more efficient than simple dilution refolding. Finally, thiol-modified PS-grafted-PNIPAM particles with different concentrations of sulphydryl groups were synthesized by RAFT polymerization. Studies on refolding of lysozyme assisted by thiol-modified PS-grafted-PNIPAM particles were carried out for further application.Modified PNIPAM hydrogels assisted protein refolding via the universal hydrophobic interactions between polymers and protein molecules. They could highly increase the refolding efficiency with the advantages of easy handling, recycling and low cost, showing potential application in genetically engineered proteins refolding in vitro.