【作者】 赵晓丽；

【导师】 崔中利；

【作者基本信息】 南京农业大学 ， 微生物学， 2010， 博士

【摘要】 表达载体的构建是实现高效表达的关键步骤,而作为表达载体组成元件之一的启动子是非常重要的。理想的启动子应具有以下特性：作用强,严格调控,易转导入其他E.coli以便筛选大量的用于生产蛋白的菌株,而且对其诱导是简便和廉价的。在大肠杆菌表达系统中,大部分表达载体采用诱导型启动子控制目的基因的表达。诱导型启动子能控制基因在特定时期、特定部位表达,这既避免了目的基因在宿主细胞内高表达对宿主细胞生长的影响,又可减少菌体蛋白酶对目标产物的降解。目前在外源基因表达中使用的大部分大肠杆菌启动子均来自相应操纵元,它们都带有可与阻遏蛋白特异性结合的操纵子区域。来源于大肠杆菌且有着超强能力的rRNA P1启动子是由包括-10区和-35区的核心启动子、一个顺式作用元件(UP元件)和3到5个反式作用转录因子(Fis)的结合位点组成的,但它们仍未被用于E.coli进行高水平表达,其主要原因是难以对其进行调控。本研究的意义就在于通过可行的方式实现对rRNA P1启动子的调控。本文首先利用大肠杆菌阿拉伯糖操纵元通过DNA环实现调控转录的特点,应用DNA重组技术构建嵌合启动子。以此启动子为基础构建了表达载体pRBB。以p-1,4-葡聚糖内切酶基因cel5G作为报告基因证实该重组表达载体可以接近pET系列商业化载体的效率进行表达。但宿主菌中的AraC调节蛋白不足以抑制启动子的转录,因此,另外构建了调节基因araC的重组质粒,通过双质粒共转化实现了对启动子的调控。同一宿主菌中的不相容性双质粒在传代中存在着分配上的不均一性,这在一定程度上影响了表达的稳定性。为了消除这一影响,同时为了更进一步验证DNA环的调控作用,我们利用大肠杆菌乳糖操纵元调控转录的特点,应用DNA重组技术构建两种嵌合启动子。其中一种是含rRNA rrnB P1启动子序列和两个lacO操纵子的嵌合启动子,分别位于核心启动子-10区下游和Fis结合位点上游,且两个lacO操纵子之间的距离是92 bp,当有阻遏蛋白(LacI)存在的情况下,它能同时与两个操纵子结合从而形成DNA环,以此来调控启动子的转录。另外一种是含rRNA rrnB P1启动子序列和一个位于核心启动子-10区下游的lacO操纵子的嵌合启动子,以此作为对照比较DNA环的调控作用。分别以这两个启动子为基础构建了不同的表达载体pRNP2和pRNP1。以本实验室克隆得到的β-1,4-葡聚糖内切酶基因cel5G作为报告基因,验证两种嵌合启动子的调控效果。结果发现只有一个lacO操纵子的嵌合启动子在没有诱导物(IPTG)的情况下已有很高的本底表达,而有两个lacO操纵子的嵌合启动子在没有诱导物存在的时候几乎没有表达外源蛋白,当加入IPTG后,外源基因可以很好地表达。另外还利用解淀粉芽孢杆菌FZB42的解淀粉芽孢杆菌核糖核酸酶barnase基因构建了pRNP2-barnase重组表达载体,再次验证了pRNP2载体在BL21(DE3)宿主菌中的严谨性。结果表明我们成功构建了基于rrnB P1启动子和DNA环调控元件的可控表达载体,此载体可以利用多种大肠杆菌作为宿主菌表达外源基因,并且在BL21(DE3)中的严谨性最佳,这对于表达有毒蛋白非常有用。同时根据载体的特性扩展了该表达载体在定向突变方面的应用。采用易错PCR体外定向进化技术,利用表达载体pRBB建立了来源于Flavobacterium sp.的去除信号肽的乙基对硫磷水解酶基因opd突变体库,并通过回复突变研究了突变位点与Oph结构间的关系。采用pRBB载体,PCR产物可直接与表达载体连接,进行目的基因的表达,省去了亚克隆的步骤,使筛选工作更加简便、快速。为了分析不同氨基酸位点对乙基对硫磷水解酶活性的影响,我们选取了2个活性丧失的突变体,每个突变体分别有3个不同的氨基酸突变位点,通过对酶分子结构的分析,发现大部分突变位点氨基酸残基位于结构表面且远离活性中心。通过重叠延伸PCR法分别构建了12种单位点和双位点回复突变酶。通过检测突变酶的活性发现,F216L和M293V的单个位点突变对乙基对硫磷水解酶活性没有影响,而当两个位点同时突变时酶活降低了50%。这一现象表明,在突变酶F216L和M293V中可能存在两个突变位点的协同叠加作用而降低了酶的催化效率,而不是单个氨基酸残基突变产生的作用。V316E的突变使酶活降低了约70%；E115G和R275H的单位点突变都使得酶活降低了近80%；198T的单突变以及R275H和V316E的双位点突变都使得酶活全部丧失。这些结果也揭示了处于非活性中心或非结合位点的突变位点可能通过改变整个酶分子的空间位阻、催化残基之间的氢键网络形成来改变酶的催化特性。本研究为采用定向进化的方法改造有机磷水解酶提供了有价值的理论参考。更多还原

【Abstract】 The construction of an expression vector requires several elements whose configuration must be carefully considered to ensure the highest levels of protein synthesis. The value of expression vector based on useful promoter is well recognized in modern biotechnology. A useful promoter exhibits several desirable features:it is strong, has a low basal expression level, easily transferable to other E. coli strains to screen a large number of strains for yielding protein, and its induction is simple and cost-effective. In E. coli expression system, the expression of target gene was regulated by inducible promoters in most expression vectors. Inducible promoters can control gene expression at particular time and specific parts, which not only avoids effect on growth of host cells when target genes were expressed in them, but also reduces the proteolysis of the target products. At present, most E.coli promoter used in expression of heterologous gene are derived from the corresponding operon All of them harbor operon region, which could be binded specifically with repressor proteins.The high-activity rrnB P1 promoter derived from E. coli contains a core promoter, a cis-acting DNA sequence (the UP element), and 3-5 trans-acting transcription factor (Fis) binding sites. Although the extraordinary strength of the rRNA promoters P1 and P2 is well documented, these promoters have not been exploited for the high-level expression of proteins in E. coli, mainly because of their difficult regulation. The significance of this study is to achieve regulation of 16rRNA P1 promoter.In this study, based on loop-controlled regulation system of the arabinose operon and rRNA rrnB P1 promoter, one chimeric promoter was constructed using gene recombinant techniques. Based on the promoter, an expression vector pRBB was constructed. A recombinant expression vector pRBB-cel5G was constructed usingβ-1,4-glucanase gene cel5G as a reporter and transformed into E.coli TOP 10. Based on comparison of expression with or without induction of iducer (arabinose), we found that the AraC regulatory protein in host strains couldn’t repress the transcription. Therefore, another recombinant expression plasmid harboring regulatory gene araC was also constructed. And we could regulate the promoter by co-transforming both of the two plasmids.In order to eliminate the effect of the instability of expression, and prove the regulation of DNA loop, based on loop-controlled regulation system of the lactose utilization operon and rRNA rrnB P1 promoter, two chimeric promoters were constructed using gene recombinant techniques. One chimeric promoter contains rRNA rrnB P1 promoter sequence and two lacO sites located 92bp apart, one of which was located downstream of the -10 site and the other was located upstream of the Fis site. When a tetrameric lac repressor (LacI) binds to two lacO sites without the presence of inducer (IPTG), a DNA loop is formed that could repress the transcription of the heterologous gene. There was only one lacO in another promoter which was located downstream of the -10 site and this promoter was designed as a control to compare the regulative effects of looping. Based on the two different promoters, two expression vectors were constructed and designated as pRNP2 and pRNP1 respectively. The efficiency of the rrnB P1 expression system was evaluated using the endo-β-1,4-glucanase gene as a reporter. The results indicated that the regulation of promoter by DNA looping significantly increased the repression level. DNA looping could eliminate most of the undesired basal transcription under non-inducing conditions. To verify the stringent regulation of promoter by the loop structure, we expressed ribonuclease barnase gene of Bacillus amyloliquefaciens in E. coli BL21 (DE3) using pRNP2-barnase expression vector. The results indicated that the barnase gene was stringently regulated in E. coli BL21 (DE3). All these results proved that we have developed a high-level expression system based on the rRNA rrnB P1 promoter and loop regulation strategy. The expression vector allowed a higher expression level of recombinant protein in different E. coli host strains. Furthermore, the vector was a tightly regulated expression vector, especially in the E. coli host strain BL21 (DE3). This feature might be useful for the expression of toxic proteins in E. coli.An ethyl parathion hydrolase opd gene mutant library was established by directed evolution techniques of error-prone PCR using the expression vector pRBB. Expression vector pRBB could be ligated with PCR products directly to express target protein, so it made screening more convenient and fast. In order to analyze the effect of different amino acid positions towars ethyl parathion hydrolase activity, two Oph mutants losing activity were selected from the mutant library, both of which had three site mutations. Structural models of two mutant proteins revealed that most of the mutation points of amino acid residues were located in the surface of the structure, and far away from catalytic center of the enzyme.For further study of the functions of substitutions in the mutants,12 site-mutation variants were constructed by using SOE-PCR. By detecting the activities of the mutants, substitutions F216L and M293V with single mutation site were found to show slight decreased activity compared with wild type enzyme, whereas simultaneous mutation with the same two sites results in reduction to 50% of original enzyme. This phenomenon indicated that the decreased activity of double sites mutant was not due to the contribution of the single substitution, but due to the synergistic effect of multi-site substitutions. V316E substitution caused a reduction of 70% of the original enzyme activity; E115G and R275H mutations had made the activity decreased by nearly 80%;I98T single site mutation and R275H and V316E double sites mutation lost all of the enzyme activity. All of the results revealed that mutation sites outside of the catalytic center or the binding sites changed catalytic activity by changing formation of hydrogen bonds of catalytic residues in active site. This study provided useful references for directed evolution of the ethyl parathion hydrolase.更多还原