【作者】 葛翔；

【导师】 姚善泾； 关怡新；

【作者基本信息】 浙江大学 ， 生物化工， 2009， 博士

【摘要】 目前,蛋白质体外重折叠复性技术已成为基因工程蛋白质产业化的瓶颈问题之一。蛋白质体外重折叠过程中聚集体的形成是制约蛋白质恢复正确空间结构的一个主要原因,向复性体系中加入能够协助目标蛋白复性的试剂,即折叠助剂,是解决这一问题的有效方法,因而开发新型、高效,同时又易于操作和回收利用的蛋白质体外复性添加剂成为近年来的研究热点。N-异丙基丙烯酰胺(NIPA)类聚合物是应用广泛的一种温敏型聚合物,是一种蛋白质体外复性的候选添加剂。本文以NIPA类聚合物的合成、性能表征及其在协助蛋白质体外复性中的应用为主线,开展了四个方面的工作:其一,交联网状聚合物PNIPA、NIPA与丙烯酸钠(SA)的网状共聚物P(NIPA-co-SA)的合成、性能表征及其协助目标蛋白溶菌酶(lysozyme)体外复性效果的考察;其二,线性聚合物PNIPAAm的合成、性能表征及其协助溶菌酶体外复性效果的考察;其三,兼具交联网状聚合物和线性聚合物优点的接枝型聚合物—含温敏性PNIPAAm毛发的聚苯乙烯微球(PS-grafted-PNIPAAm)的制备、性能表征及其协助溶菌酶体外复性效果的考察;其四,三种不同类型聚合物温敏性能及其协助蛋白质体外复性效果的比较,并对其协助复性的机理进行了探讨。交联网状聚合物的合成、性能表征及其协助溶菌酶体外复性效果的考察:采用反相悬浮聚合法合成了粒状PNIPA凝胶及其与丙烯酸钠(SA)的共聚凝胶P(NIPA-co-SA),温敏性能研究表明,两种凝胶具有相似的温度敏感性,但其低临界溶解温度(LCST)有所不同,分别为33℃和39℃;凝胶粒子的直径在200～500μm之间,表面分布着不规则微孔。在温度为30℃,尿素浓度为3mol/L,GSH和GSSG的浓度分别为3mmol/L和0.375mmol/L(GSH:GSSG=8:1)的复性条件下,当目标蛋白溶茵酶的终浓度为250μg/ml时,加入2mg/ml PNIPA粒状凝胶可使溶菌酶的活力回收率达到72.9%,明显高于稀释复性的51.3%;加入3mg/ml粒状P(NIPA-co-SA)共聚凝胶可使溶菌酶的活力回收率提高到71.5%。不同稀释倍数下两种凝胶协助复性实验表明,溶菌酶初始浓度越高,与稀释复性相比凝胶协助复性的效果就越好,在蛋白质体外重折叠过程中具有一定的优势。两种凝胶重复利用6次后溶菌酶的活力回收率仍分别高出稀释复性15%和12%以上。两种凝胶协助复性和稀释复性的时间基本相同,凝胶的加入并没有成为复性的限速步骤。线性聚合物的合成、性能表征及其协助溶菌酶体外复性效果的考察:采用自由基聚合法,以偶氮二异丁腈(AIBN)为引发剂,N-异丙基丙烯酰胺(NIPA)为单体,在乙醇溶液中合成了PNIPAAm。温敏性能研究表明,其低临界溶解温度(LCST)约为32℃,该值受PNIPAAm基团的亲水疏水相互作用强弱变化的控制,随PNIPAAm分子量、溶液浓度和溶液组成不同而略有变化。PNIPAAm凝胶具有较好协助溶菌酶复性的效果,与稀释复性相比,PNIPAAm可大大提高蛋白复性的初始浓度,在协助蛋白质复性上有较大优势。当溶菌酶浓度为600μg/ml时,活性回收率从稀释复性的11.93%提高到79.83%,改进非常明显。系统考察了复性液中PNIPAAm与溶菌酶浓度比(w/w)、尿素浓度和复性温度对溶菌酶复性效果的影响,确定本实验中合适的复性条件为,PNIPAAm与溶菌酶浓度比8、尿素浓度1.8mol/L、复性温度30℃。接枝型聚合物PS-grafted-PNIPAAm微球的制备、性能表征及其协助溶菌酶体外复性效果的考察:采用无皂乳液聚合法和紫外光引发表面接枝技术制备了PS-grafted-PNIPAAm微球,获得了Particle A(PS核心直径442nm,PNIPAAm高分子毛发层厚度122nm)和Particle B(PS核心直径442nm,PNIPAAm高分子毛发层厚度89nm)两种微球。微球形貌为圆球形,且粒径分布较为均一,单分散性良好,尺寸为亚微米级,其LCST均在34℃左右。将PS-grafted-PNIPAAm微球应用于溶菌酶的体外复性中,优化的复性工艺条件为:复性温度30℃,复性液中尿素浓度3mol/L,GSH与GSSG的浓度比8:1(其中GSSG的浓度0.375mmol/L),Particle A和Particle B与溶菌酶的浓度比分别为0.5和1.0。在优化的复性条件下,Particle A和Particle B的加入可以在较高蛋白浓度条件下显著地提高其活性收率,当溶菌酶的浓度为500μg/ml时,Particle A和Particle B的加入使得复性后的活性回收率分别达到了71.5%和58.0%,比稀释复性的收率提高了36.7和23.2个百分点,从而极大地降低了复性过程的成本。Particle A和ParticleB的加入,在提高溶菌酶活性收率的同时,使复性过程达到平衡所需的时间略有延长,从稀释复性时约4 h延长至4.5 h。三种聚合物温敏性能、协助蛋白质体外复性效果的比较,及其协助复性的机理初探:在上述实验的基础上,将制得的三种聚合物进行性能及协助复性效果的比较。三种聚合物都具有显著的温度敏感性,但低临界溶解温度(LCST)有所不同。在协助蛋白质体外复性的过程中,线性聚合物的效果最佳,但复性完成后不易与目标蛋白分离,难以重复利用;交联网状聚合物易于从复性体系中分离出来,但协助复性效果相对较差,且由于凝胶网络的吸附会造成蛋白损失;接枝型聚合物则兼具高效和易分离的特点,其应用前景值得期待。采用动力学模型拟合和荧光光谱分析的方法对N-异丙基丙烯酰胺类聚合物协助复性的机理进行了初步研究,结果表明,在复性过程中起主要作用的是其上的疏水基团—异丙基与溶菌酶疏水区域之间的疏水相互作用,这种作用阻止了蛋白折叠过程中的聚集沉淀,使复性向天然态的构象进行。总之,全文围绕N-异丙基丙烯酰胺类聚合物的制备、性能表征和在蛋白质复性中的应用开展了系列研究,成功完成了制备-性能表征-应用-机理的研究路线,为进一步开发新型、高效、易于分离回收的蛋白复性添加剂奠定了基础。更多还原

【Abstract】 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.更多还原