Protein Refolding Assisted By A Single Chain Of Poly (N-isopropylacrylamide) In Vitro

One of the vital problems in protein refolding in vitro is to reduce the intermolecular aggregatets which prevent protein to form natural conformation.To solve this problem,researchers make efforts to add some folding agents which can inhibit aggregation.Poly(N-isopropylacrylamide)(PNIPAM)is one of popular temperature sensitive polymers and has been applied successfully in many fields.A single chain of PNIPAM was synthesized using free-radical polymerization reaction.The lower critical solution temperature(LCST)of PNIPA was at about 32℃, and this value would slightly change with the molecular weight,solution concentration of PNIPAM and solution compositions.PNIPAM was firstly applied into the refolding of model protein lysozyme in vitro.Compared with simple dilution refolding,renaturation of lysozyme assisted by PNIPAM showed many advantages.When initial lysozyme concentration was as high as 600μg/mL,the activity was increased from 11.93%to 79.83%with the presence of PNIPAM.Some important process parameters,such as initial lysozyme concentration, the concentration ratio of PNIPAM to lysozyme,urea concentration and refolding temperature were investigated in detail and the optimized refolding condition was ascertained:the concentration ratio(w/w)of PNIPAM to lysozyme 8,urea concentration 1.8mol/L,and refolding temperature 30℃.Considering the change of fluorescence emission spectrum of lysozyme during refolding,it was assumed that the hydrophobic interaction between isopropyl of PNIPAM and refolding intermediates of protein played an important role in the process.This hydrophobic interaction between PNIPAM and protein molecules inhibited intermolecular hydrophobic interaction of protein molecules and then reduced aggregation of refolding intermediates,so the protein could be folded correctly to native conformation.Based upon the mechanism on lysozyme refolding assisted by PNIPAM,some preliminary researches on the refolding of recombinant human interferon gamma (rhIFN-γ)expressed as inclusion body were carried out.The results indicated that PNIPAM could indeed assist rhIFN-γrefolding and was more effective than simple dilution refolding.When initial concentration of rhIFN-γwas 50μg/mL,the final active concentration could be up to 27μg/mL in the presence of PNIPAM,while the final active concentration was only 5μg/mL in dilution refolding.The optimal refolding condition of rhIFN-γin this experiment was different from that of lysozyme, the concentration ratio of PNIPAM to rhIFN-γwas 20 and refolding temperature was 30℃.PNIPAM assists protein refolding via the universal hydrophobic interaction,so it can be applied to a large number of genetically engineered proteins.Forthermore,it was an efficient,low-cost and convenient method.