The Preparation Of Poly(N-isopropylacrylamide) Thermo-sensitive Polymers And Its Application In Assisting Protein Refolding In Vitro

Nowadays,how to refold proteins expressed as inclusion bodies into natural conformation with high yields at high initial proteins concentration is becoming one of the most important problems in genetic engineered proteins industry.The vital step in protein refolding in vitro is to reduce the intermolecular aggregates which prevent protein to form correct three-dimensional structure.Some folding agents which can inhibit proteins aggregation were added to solve this problem.Therefore,the development of the novel,highly efficient,easy to operate and recycle folding agents has become a research hotspot in recent years.Poly(N-isopropylacrylamide) is one of the most popular temperature-sensitive polymers and has been widely applied in many fields.In this thesis,the different kinds of NIPA polymers,including crosslinked network polymers(PNIPA and P(NIPA-co-SA)),a single chain of polymer(PNIPAAm) and graft copolymer (PS-grafted-PNIPAAm),were synthesized.The performance of the above polymers was characterized and then the polymers were applied to the refolding of denatured hen egg white lysozyme(HEWL) in vitro.Furthermore,the assisting refolding effects of different polymers were compared and the refolding mechanism was discussed.Firstly,the PNIPA and P(NIPA-co-SA) gel particles were synthesized by inverse suspension polymerization.The two gel particles had similar temperature sensitivity but different low critical solution temperature(LCST).The LCST of the gels was 33℃and 39℃respectively.The gel particles were characterized by SEM and the results showed that the particles were in the range of 200-500μm in diameters with numerous pores spreading over the surface of the beads.The optimum condition for lysozyme refolding assisted by PNIPA and P(NIPA-co-SA) gel particles was obtained, i.e.refolding temperature 30℃,the concentration of urea 3mol/L and the concentration ratio of GSH to GSSG 8:1(the GSSG concentration 0.375mmol/L). Under this optimized condition,when the final protein concentration was 250μg/ml, the activity recovery of lysozyme increased from 51.3%to 72.9%assisted by PNIPA gel particles of 2mg/ml concentration,while to the 71.5%assisted by the P(NIPA-co-SA) gel particles of 3mg/ml concentration correspondingly.The results indicated that gel particles were even more efficient at high protein concentration. After PNIPA and P(NIPA-co-SA) gel particles were recycled for 6 batches,the activity recovery of lysozyme were still 15%and 12%higher than the simple dilution. The refolding time mediated by the two kinds of gel particles was nearly the same with the simple dilution.Secondly,a single chain of PNIPAAm was synthesized using free-radical polymerization reaction.The results of temperature sensitivity experiments showed that the LCST of PNIPAAm was at about 32℃,and this value would slightly change with the molecular weight,solution concentration of PNIPAAm and solution compositions.Compared with the simple dilution refolding,the renaturation of lysozyme assisted by PNIPAAm showed some advantages.When lysozyme concentration was as high as 600μg/ml,the activity was increased from 11.93%to 79.83%with the presence of PNIPAAm.Some important process parameters,such as the initial lysozyme concentration,the concentration ratio of PNIPAAm to lysozyme, the urea concentration and the refolding temperature were investigated in detail and the optimized refolding condition was ascertained:the concentration ratio(w/w) of PNIPAM to lysozyme 8,the urea concentration 1.8mol/L,and the refolding temperature 30℃.Thirdly,PS-grafted-PNIPAAm Particle A(diameter of PS core 442nm,thickness of brush layer 122nm) and Particle B(diameter of PS core 442nm,thickness of brush layer 89nm) were synthesized by the non-soap emulsion polymerization and UV-initiation.The particles were in spherical shape with narrow particles size distribution and good dispersion property.The LCST was about 34℃.The optimum condition for lysozyme refolding mediated by PS-grafted-PNIPAAm was:the refolding temperature 30℃,the concentration of urea 3mol/L and the concentration ratio of GSH to GSSG 8:1(the GSSG concentration 0.375mmol/L),and Particle A and B to lysozyme were 0.5 and 1 respectively.Under this condition,lysozyme was refolded efficiently assisted by Particle A and B,especially at high initial protein concentrations.For instance,when lysozyme concentration was 500μg/ml,the activity recovery assisted by Particle A and B obtained 71.5%and 58.0%respectively,36.7 percent and 23.2 percent higher than simple dilution.Meanwhile with the additive Particle A and B,the refolding time was extended from 4h to 4.5h,compared with simple dilution refolding.Fourthly,based on the experimental results,the comparison of performance and the assisting refolding effects of different polymers were carried out.Three kinds of polymers all had distinct temperature sensitivity,but the LCST were a little bit different.In the process of assisting refolding,the linear PNIPAAm was the most effective but hard to be removed and recycled after protein refolding;the crosslinked network polymers were easily to be removed from the refolding system but the efficiency was correspondingly lower and there was loss of protein due to the penetration and adsorption of protein into the gel network;the graft copolymer possessed both advantages of liner and crosslinked network polymers with high efficient and easily to be separated.The preliminary studies to the mechanism of assisting refolding with kinetic model simulation and fluorescence spectrum analysis were carried out,and the results showed that the hydrophobic interaction between NIPA polymers 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.In a word,the preparation,characterization and application of NIPA polymers were carried out.Some important information was obtained and the refolding mechanism was discussed,which would certainly be useful for the development of some novel,highly efficient,easy to separate and recycle folding agents.