【作者】 王君；

【导师】 刘铮；

【作者基本信息】 清华大学 ， 化学工程与技术， 2004， 博士

【摘要】 蛋白质复性技术的研究对于基因工程重组蛋白质的生产具有重要意义。本论文以天然溶菌酶为模拟体系,综合运用实验研究、结构分析和分子模拟等方法研究表面活性剂十六烷基三甲基溴化铵(以下简称CTAB)辅助溶菌酶复性过程的热力学和动力学特性。在此基础上,以重组人溶菌酶与重组甘露聚糖酶为实际体系,通过实验研究CTAB及其它类型折叠助剂的辅助复性效果,以考察此类技术的适用范围。第一章对蛋白质复性技术及其研究现状进行了简要综述。本章首先介绍了蛋白质复性技术研究所涉及的基本理论和实验检测方法;评述了目前发展的各种复性技术及其在基础研究和实际应用方面存在的问题;总结了表面活性剂辅助蛋白质复性技术的研究成果并重点分析了此类技术研究中存在的问题。继而提出了本文的研究设想,即从复性中间产物形成过程的分析入手来认识复性过程的热力学和动力学特性;将活性分析、结构测定和分子模拟等方法相结合来研究复性过程微观机理;采用不同的重组蛋白质产品进行复性实验以考察复性技术的适用性。以此为基础确立了本文研究工作的目标和内容框架。第二章通过实验研究发现在复性液中加入低浓度CTAB能够有效地提高溶菌酶的复性率,当CTAB与变性溶菌酶摩尔比为10时复性效果最佳。溶液表面张力与酶活力的变化显示变性溶菌酶首先与CTAB快速形成复合物,进而在氧化/还原剂作用下开始复性并缓慢与CTAB发生解离,圆二色性光谱与荧光光谱分析表明CTAB在与变性溶菌酶形成复合物时帮助其形成丰富的二级结构而促进折叠;但若结合CTAB过多则会阻碍折叠过程进行。非还原型SDS-PAGE、离子交换色谱等技术的分析结果表明CTAB辅助复性的主要产物包括具有活性的溶菌酶、无活性的溶菌酶多聚体及溶菌酶单体,产物的分布取决于溶液中CTAB与溶菌酶的摩尔比。以上实验结果未见国内外文献报道。第三章通过实验考察了CTAB与β-环糊精(以下简称β-CD)构成的人工分子伴侣系统辅助溶菌酶复性过程。对不同类型表面活性剂的考察结果表<WP=5>明表面活性剂电荷性质对于辅助效果影响显著。本章对人工分子伴侣复性操作流程进行了改进,显著降低了复性所需CTAB及β-CD的用量,同时提高了复性率。非还原性SDS-PAGE证实复性产物为活性溶菌酶单体与聚集体沉淀,其组成与CTAB用量无关。通过考察β-CD对CTAB-溶菌酶复合物的影响证实CTAB与变性溶菌酶可形成不同组成的复合物,复合物的分布随时间而变。在此基础上提出分步加入β-CD的复性操作方法,显著地提高了蛋白质在较高浓度下的复性率。第四章中建立了CTAB辅助复性的宏观动力学模型,并通过动力学实验拟合得到模型参数。在本文研究范围内模型计算结果与实验结果具有很好的一致性:CTAB浓度的增加导致复性速率下降、复性率下降。模型分析结果指出复性体系中去除CTAB有利于提高复性速率及复性率,但由此可能导致CTAB-变性溶菌酶复合物解离速率常数增大,造成聚集体生成过多而不利于复性。综合上述研究结果提出了CTAB辅助溶菌酶复性过程的机理模型,CTAB-变性溶菌酶复合物的解离为复性过程中的速度控制步骤。第五章采用动态Mante Carlo模拟来研究表面活性剂辅助蛋白质折叠的热力学特性。分子构建采用HP模型,分子间的相互作用采用方阱类势函数。模拟结果显示:模型蛋白分子折叠过程中可能陷入某些局部能量最低构象的中间态而使得折叠过程被阻止;加入折叠助剂可导致变性蛋白质形成丰富的中间态,使折叠过程中的能量阱曲面变得光滑,从而推动蛋白质完成折叠。通过模拟考察了表面活性剂的疏水性及浓度对辅助折叠行为的影响,模拟结果与本文结果及已报道的实验结果一致。第六章采用重组人溶菌酶与重组甘露聚糖酶等实际体系,考察了CTAB、人工分子伴侣及其它折叠助剂的辅助复性效果。实验结果显示:表面活性剂辅助复性技术更适于辅助那些易形成聚集体的重组蛋白质的复性;氨基酸残基序列及高级结构相似的蛋白质其复性过程特性也相似。此外,包涵体的纯化对于重组蛋白质的最终复性效果有重要影响。更多还原

【Abstract】 The research into protein refolding techniques is of essential importance to the down stream processing of genetically engineered proteins. The experimental studies in this dissertation have shown an effective refolding of denatured lysozyme by dilution using the refolding buffer containing low concentration cetyltrimethyl- ammonium bromide (CTAB) and indicated its high potential in large scale processing. This dissertation was devoted to the comprehensive understanding of the process fundamentals of this novel refolding operation, using native hen egg white lysozyme as the protein sample. The interaction between protein and surfactant, the key to the formation and the dissociation of protein-surfactant complex, was investigated using various experimental and analytical procedures, as well as molecular simulation using simple lattice model with dynamic Monte Carlo simulation. The feasibility of this new refolding method was examined through its application to the refolding of recombinant human lysozyme and recombinant β-mannanase, in which other refolding methods were also tested. Chapter 1 started with a brief overview of the theoretical and experimental studies of protein refolding. Then a detailed summary of the refolding techniques was presented, in which the development of surfactant assisted protein refolding methods was highlighted due to its proven potential in large scale refolding. The problems and prospects of the research in this field were discussed. It was concluded that the formation and dissociation of protein-surfactant complex was the key to an effective refolding. It was thus the focus of studies on the refolding process fundamentals in terms of thermodynamics and kinetics. The latter should be based on both experimental studies using different protein samples and theoretical investigations centered on the molecular interaction between surfactant and protein and its implementations to protein structure. At the end of this chapter, a framework <WP=7>of the study presented in this dissertation was illustrated. Chapter 2 investigated the refolding of lysozyme by dilution with the refolding buffer containing low concentration CTAB. It was reported for the first time that the addition of CTAB in the refolding buffer led to a substantial increase in the refolding. The refolding yield was a function of the molar ratio of CTAB to lysozyme and the maximum yield was obtained at a ratio of 10. The changes of the surface tension of refolding solution indicated a rapid formation of CTAB-denatured lysozyme complex and a slow dissociation, upon the addition of redox reagents, i.e., GSSG/GSH, which led to the refolding to native lysozyme. The circular dichroism spectra analysis indicated the addition of CTAB led to enriched native-like secondary structures of lysoyzme, which favored the refolding. However the refolding was hindered once too many CTAB molecules were involved into the complex. Analysis of the refolding products by non-reductive SDS-PAGE and ion exchange chromatography showed the existence of native lysozyme, soluble aggregates and the CTAB-denatured lysozyme as major products in CTAB assisted refolding and the distribution of these products were determined by the molar ratio of CTAB to the denatured lysozyme. Chapter 3 presented the refolding of lysozyme using surfactant and β-cyclodextrin (β-CD), an established method known as artificial chaperone. It was shown that the charge properties of surfactant affected the refolding performance significantly. In case of CTAB and β-CD being used, the refolding was observed again as a function of the molar ratio of CTAB to denatured lysozyme and the optimal refolding was obtained at 10. A new operation scheme was developed in the present study, which greatly increased the refolding through the effective inhibition of the formation of protein aggregates and reduced the consumption of surfactant and β-CD. Analysis by SDS-PAGE showed the native lysozyme and insoluble aggregates as the major <WP=8>products of the refolding and the dist更多还原