【摘要】 强信号肽的使用是提高蛋白分泌效率的关键,筛选强信号肽具有重要的应用价值。本试验以大肠杆菌-谷氨酸棒杆菌基础穿梭载体pAU2为骨架,以枯草芽孢杆菌的碱性丝氨酸蛋白酶AprE为报告蛋白,分别使用强启动子tac-M和谷氨酸棒杆菌核糖体结合位点一致序列来控制报告基因的转录和翻译,成功构建谷氨酸棒杆菌信号肽探测载体pAU20。分别对来自枯草芽孢杆菌、地衣芽孢杆菌、以及谷氨酸棒杆菌中共16种Sec型信号肽编码序列进行PCR扩增,与pAU20连接,转化谷氨酸棒状杆菌,获得重组信号肽探测载体工程菌株。工程菌株摇瓶发酵培养物上清液AprE酶活测定结果表明,C. glutamicum/pAU20-Vpr、C. glutamicum/pAU20-BprA上清液比酶活分别为180.02和167.6U/mg蛋白质,均显著高于C.glutamicum/pAU20-cgr2070上清液比酶活(105.09 U/mg蛋白质);C. glutamicum/pAU20-oppA、C. glutamicum/pAU20-Epr、C. glutamicum/pAU20-cgr2063和C.glutamicum/pAU20-ywaD上清液比酶活分别为96.68、86.18、82.52和80.56U/mg蛋白质,均稍低于C. glutamicum/pAU20-cgr2070上清液比酶活。这些结果表明,信号肽Vpr、BprA、cgr2070、oppA、Epr、cgr2063和ywaD都属于谷氨酸棒杆菌强信号肽。更多还原

【Abstract】 The use of strong signal peptides is the key to improve the secretion efficiency of proteins, and the screening of strong signal peptides is of importance. In this study, the signal peptide-probe vector pAU20 was successfully constructed using the basic E. coli-C. glutamicum shuttle vector pAU2 as the backbone, and the alkaline serine protease AprE of Bacillus subtilis was used as the reporter protein. The strong promoter tac-M was used to control aprE transcription, and the consistent sequence of the C. glutamicum ribosome binding site was used to control AprE translation. 16 Sec-type signal peptide-encoding sequences from B. subtilis, Bacillus licheniformis, and C. glutamicum were amplified using PCR technology, ligated to the vector pAU20, and transformed into the competent cells for generating the engineered C. glutammicum strains harboring the recombinant signal peptide-probe vectors. The test of AprE activities in the supernatants of the engineered C. glutamicum strain cultures demonstrated that the specific activities of the C. glutamicum/pAU20-Vpr and C. glutamicum/pAU20-BprA strains were 180.02 U/mg protein and 167.6 U/mg protein, respectively, which were significantly stronger than that of C. glutamicum/pAU20-cgr2070(105.09 U/mg protein). The specific activities of C. glutamicum/pAU20-oppA, C. glutamicum/pAU20-Epr, C. glutamicum/pAU20-cgr2063 and C. glutamicum/pAU20-ywaD were 96.68, 86.18, 82.52 and 80.56 U/mg protein, respectively, which were slightly weaker than that of C. glutamicum/pAU20-cgr2070. These results demonstrated that Vpr, BprA, cgr2070, oppA, Epr, cgr2063 and ywaD are strong signal peptides in C. glutamicum.更多还原