| 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. |
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