重组大肠杆菌表达包涵体药物蛋白的复性与纯化研究

发布时间：2018-06-07 14:33

本文选题：大肠杆菌 + 药物蛋白　；参考：《华东理工大学》2017年硕士论文

【摘要】：大肠杆菌表达系统是目前应用最为广泛的重组蛋白表达系统。由于蛋白表达速度过快、活性二硫键来不及正确配对等等原因,外源蛋白在大肠杆菌胞内往往会以无活性的包涵体形式表达。由于包涵体复性折叠机制不明确,把无活性的包涵体复性为完全的天然活性状态是一件很难的事情。较低的复性效率也仍然是大肠杆菌体系表达的许多外源蛋白造成损失或者难以有效制备的主要原因。本课题以重组人表皮生长因子(hEGF)和新型HIV疫苗蛋白为研究对象,研究在包涵体药物蛋白的复性纯化过程中如何根据具体目的和要求,决定复性与纯化的先后顺序、选择复性方法和纯化方法、合理设计纯化方案(合理衔接不同的纯化方法)等等,以期为其他包涵体药物蛋白的复性纯化提供一定的参考价值。非融合表达hEGF蛋白的复性纯化研究:采用的是"先复性后纯化"的策略。经过不断探索,最终确定hEGF蛋白的复性纯化工艺为:包涵体洗涤→包涵体变、复性→50%硫铵沉淀→凝胶过滤层析→DEAE阴离子交换层析。hEGF纯化样品的SDS-PAGE电泳纯度为97%,HPLC液相色谱纯度为96%;蛋白纯化回收率为18%;且此纯化样品对Balb/c3T3细胞(小鼠胚胎成纤维细胞)具有明显的生长促进作用。新型的药物蛋白—HIV辅助T细胞疫苗的复性纯化研究:(1)HIV疫苗包涵体蛋白的变性条件优化。包涵体蛋白先用7M盐酸胍变性缓冲液变性溶解,接着用8M尿素缓冲液对其进行稀释,使盐酸胍浓度降至1M。如此变性处理后的包涵体蛋白结构更趋于稳定,后期纯化及复性过程中都不会发生蛋白沉聚现象。(2)HIV疫苗包涵体蛋白的纯化与复性。选择"先纯化后复性"的策略,研究出一套成熟的复性纯化工艺:包涵体洗涤→包涵体变性→镍柱亲和层析(HisTrapFF)→ 30%硫铵沉淀→CMFF阳离子交换层析→镍柱亲和层析(HisTrap HP)。最后目的蛋白的电泳纯度提高到95%。HIV疫苗包涵体蛋白的纯化总回收率为13%。纯化后的变性状态的包涵体蛋白复性方法选择透析复性,复性过程中没有产生明显的蛋白沉淀。HIV疫苗蛋白的成功复性分离纯化为进一步研究HIV辅助T细胞疫苗蛋白的理化性质以及生物学功能提供了保障。
[Abstract]:E. coli expression system is the most widely used recombinant protein expression system. Due to the rapid expression of proteins and the late matching of active disulfide bonds, foreign proteins are often expressed in the form of inactive inclusion bodies in E. coli cells. Because the folding mechanism of inclusion body is not clear, it is difficult to renaturation the inactive inclusion body into a completely natural active state. Low renaturation efficiency is still the main reason for the loss of many foreign proteins expressed in Escherichia coli system or the difficulty of effective preparation. In this paper, recombinant human epidermal growth factor hEGF) and novel HIV vaccine proteins were used to study how to determine the sequence of renaturation and purification according to specific objectives and requirements in the renaturation and purification of inclusion body drug proteins. The methods of renaturation and purification were selected, and the purification schemes were designed reasonably (linking up with different purification methods) so as to provide some reference value for the renaturation and purification of other inclusion body drug proteins. Renaturation and purification of non-fusion expressed hEGF protein: the strategy of renaturation and purification was adopted. After continuous exploration, the final process of renaturation and purification of hEGF protein was as follows: inclusion body washing, inclusion body transformation, DEAE anion exchange chromatography. The purity of SDS-PAGE electrophoresis was 97%. The purity of HPLC was 96. The recovery rate of protein purification was 180.The purified sample was used to purify Balb/c3T3 cells (mouse embryogenesis). Fibroblasts) have obvious growth-promoting effects. Study on renaturation and purification of novel Drug protein-HIV-assisted T Cell Vaccine the denaturation conditions of inclusion body proteins of the 1: 1 HIV vaccine were optimized. The inclusion body protein was first denatured and dissolved with 7m guanidine hydrochloride denaturation buffer, then diluted with 8m urea buffer to reduce the concentration of guanidine hydrochloride to 1M. The structure of inclusion body protein after denaturation is more stable, and no protein deposition will occur during the later purification and renaturation. The purification and renaturation of inclusion body protein of HIV vaccine. By selecting the strategy of "first purification and then renaturation", a set of mature renaturation and purification techniques were developed: inclusion body washing body denatured nickel column affinity chromatography (HisTrapFF) with 30% ammonium sulfide precipitation and CMFF cation exchange chromatography and nickel column affinity chromatography (HisTrap HP). Finally, the purity of the protein was improved to 13% after purification of the inclusion body protein of 95%.HIV vaccine. The method of renaturation of inclusion body protein in purified denatured state was selected for dialysis renaturation. The successful renaturation and purification of HIV vaccine proteins without obvious protein precipitation during renaturation provided a guarantee for further study on the physicochemical properties and biological functions of HIV assisted T cell vaccine proteins.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ460.1

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