【作者】 曹丽娟；

【导师】 邹竹荣；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 蛋白质工程一直是生物技术研究的一个重要方面。在科学研究中,获得大量纯化、可溶性的、有生物活性的蛋白质是蛋白质理化性质与功能研究的基础。然而受资源有限、成本昂贵等因素的影响,天然的蛋白质无法满足实际需要。随着蛋白质工程和基因工程技术的发展,重组蛋白在理化性质和生物学活性上已日趋接近于天然蛋白质,且可以通过大规模生产源源不断地获得。虽然20世纪90年代后开始频繁利用哺乳动物细胞来生产重组蛋白,但迄今为止原核的大肠杆菌表达系统还是最常用的,因为大肠杆菌的遗传背景清楚,培养条件简单,表达高效,经济方便。但是,大肠杆菌表达系统也有局限性,当所表达的蛋白是复杂的真核来源的蛋白时,大肠杆菌常常不能将翻译出的多肽链正确折叠修饰形成蛋白质的天然构象,而是形成不溶性的无功能的包涵体。包涵体中多肽链活性恢复必须通过复杂的变性复性过程,而且成功率低。因此,探索重组蛋白在大肠杆菌中的可溶性表达具有较高的学术价值和广泛的应用前景。目前已经找到了一些提高重组蛋白在大肠杆菌中可溶性表达的方法,其中较常用的办法是使用融合表达策略,即将融合标签和靶蛋白连接成为一个融合蛋白进行表达。但由于蛋白质种类繁多,目前还没有找到可以提高蛋白可溶性表达的通用型融合标签。本工作中,我们致力于寻找一类通用的增溶标签来促进靶蛋白(尤其是那些用常规增溶办法无法解决的蛋白)在大肠杆菌中的可溶性表达。基于此,我们设计挑选了三种大肠杆菌来源、分子量相对较小和酸度超强的融合标签即Msb(大肠杆菌酸性蛋白MsyB)、Yd(一个推定的大肠杆菌开放阅读框蛋白,yjgD)和Od(大肠杆菌RNA聚合酶σ70因子rpoD的N端区);分析它们对三种具有高度聚集倾向性的靶蛋白即小牛肠激酶(EK)、烟草蚀纹病毒蛋白酶(TEV)和1,5—二磷酸核酮糖羧化/加氧酶大亚基rbcL(Rb)在大肠杆菌中可溶性表达的影响。同时,选用一些常用融合标签如大肠杆菌Trx(硫氧还蛋白)、MBP(麦芽糖结合蛋白)、NusA和酵母SUMO蛋白与它们一起进行作用比对。通过PCR或酶切得到融合标签Msb、Yd、Od、MBP、NusA、Trx和SUMO的基因,然后以质粒pET-32a(+)为骨架与靶蛋白基因融合构建一系列融合表达载体,并将它们在大肠杆菌BL21(DE3)中进行诱导表达。SDS-PAGE分析发现,与常用标签Trx等相比,三个超酸性标签蛋白Yd、Od和Msb都能够卓著地增强靶蛋白(EK、TEV和Rb)的可溶性表达。另外,我们还构建了杂合标签MS、YS、OS、NS和PS(即SUMO分别与标签Msb、Yd、Od、NusA和MBP的杂合体)与靶蛋白的融合表达载体,结果表明,酸性杂合标签MS,YS,OS也能够显著地增强靶蛋白的可溶性表达。这些实验结果很好地验证了用Wilkinson-Harrison可溶性模型得到的理论预测。进一步,我们利用酵母蛋白酶Ulp1切割SUMO融合蛋白的反应体系和TEV融合蛋白自切割反应体系评估了所选三种超酸性融合标签Msb、Yd和Od的作用机理。结果表明这些超酸性融合标签在显著增强靶蛋白可溶性表达的过程中实际上起着分子内伴侣作用,有效地帮助靶蛋白的正确结构折叠。总之,本工作发现超酸性蛋白Msb、Yd、Od可以作为一类通用高效的增溶性融合标签来促进大肠杆菌中靶蛋白的可溶性表达,相信它们在蛋白质研究及其生物技术领域内有着很好的应用前景。更多还原

【Abstract】 Protein engineering has been an important aspect in biotechnological research.In scientific area,to gain large amount of pure,soluble and bioactive protein is the prerequisite for studying its physiochemical properties and function.However, obtainment of natural protein cannot meet actual requirements,due to a few of strict limitations such as rare resources and high cost.With the development of protein engineering and genetic engineering,the recombinant proteins,being increasingly closer to their natural forms in terms of the physical,chemical and biological features, can be prepared in large scales.Although mammalian cells have been frequently applied to produce recombinant proteins since the 1990s,the Escherichia coli（E.coli） expression system is the most commonly used so far.This system is of many advantages including well-known genetic background,simple culture conditions, high-level expression,inexpensiveness and convenience.However,the E.coli expression system does have limitations.When a target protein is complicated and is of eukaryotic sources,this system often fails in making the expressed protein fold correctly,leading to formation of insoluble and non-active aggregates termed inclusion bodies.Functional recovery of proteins from their inclusion bodies usually should undergo a complex and inefficient process of denaturalization and refolding. Therefore,to maximize the soluble expression of recombinant protein in E.coli is certainly of high academic value and wide range of applications.Regarding this,some effectual approaches have been explored at present,in which the fusion expression strategy by using protein fusion partners（or tags）has been prevalently used.However,such fusion tags are not effective for all proteins.In this work,we were committed to finding common fusion partners to improve the soluble expression of target proteins（especially those recalcitrant to conventional solubilizing efforts）in E.coli.To this purpose,three candidate proteins with the features of E.coli origin,relatively small size and extremely high acidity were selected,including Msb（an E.coli acidic protein MsyB）,Yd（one hypothetical protein,yjgD）,and Od（the N-terminal domain ofσ70 factor of RNA polymerase, rpoD）.They were tested as fusion tags for three highly aggregation-prone target proteins including EK（the bovine enterokinase）,TEV（the tobacco etch virus protease） and Rb（rbcL,the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase）. Meanwhile,other frequently used solubility-enhancing fusion tags such as Trx（TrxA）, MBP,NusA and SUMO,were included for comparison.By using PCR amplification and restriction enzyme digestion,DNA fragments of fusion tags Msb,Yd,Od,MBP,NusA,Trx and SUMO were obtained and respectively fused with the genes of target proteins to generate series of expression vectors on the basis of backbone plasmid pET-32a（+）.All of these vectors were expressed in E.coli BL21（DE3）by induction and subsequently analyzed by SDS-PAGE.The results demonstrated that all three hyper-acidic protein fusion tags Yd,Od as well as Msb could remarkably enhance the soluble expression of three highly aggregation-prone target proteins Ek,TEV and Rb.In addition,hybrid fusion tags such as MS,YS,OS, PS and NS（consisting of Msb,Yd,Od,MBP,and NusA,and the common His₆-tagged SUMO,respectively）were created,the acidic ones of which were also found to effectuate apparently in solubility enhancement for target proteins.Taken together,all these experimental results are well in consistence with the theoretical solubility prediction by the revised algorithm of Wilkinson-Harrison.We further evaluated the functional roles of all three hyper-acidic fusion partners （Yd,Od and Msb）by taking advantages of the yeast SUMO/Ulpl reaction and TEV auto-cleavage.Our results indicated that these hyper-acidic fusion partners actually act as intramolecular chaperones assisting in the correct folding of the target proteins, during their solubilising process.Conclusively,the hyper-acidic proteins（Msb、Yd、Od）in our study could be used as a category of universal and potent fusion tags to improve the soluble expression of target proteins in E.coli,and largely believed to confer broad prospects in the fields of proteins studies and biotechnological applications.更多还原