大肠杆菌trpBA基因的克隆表达

Cloning and Expression of trpBA Genes from E.coli

目的:提高大肠杆菌中色氨酸合成酶的表达量和表达活性。方法:利用PCR方法从大肠杆菌K-12的基因组中直接克隆出紧密连锁trpB和trpA基因(简称trpBA),并将其连接到原核表达载体pet22b(+)中,得到重组质粒pet22b(+)-trp-BA,转化大肠杆菌BL21,IPTG诱导重组蛋白表达,表达产物经SDS-PAGE分析并用比色法测定其活性。结果:凝胶电泳可见PCR扩增产物大小约为2kb,SDS-PAGE鉴定目的蛋白的Mr分别约为29000和44000,色氨酸合成酶α、β亚基分别得到了高效表达,色氨酸合成酶活性提高到对照菌的3.7倍。结论:成功构建了重组质粒pet22b(+)-trpBA,色氨酸合成酶的表达量和表达活性在大肠杆菌中得到了提高,为高产色氨酸基因工程菌的构建奠定基础。

Objective： To increase the expression level and activity of tryptophan synthase in E.coli. Methods： The trpB and trpA genes （or trpBA genes） were amplified from E.coli K-12 by PCR method, then directly cloned into plasmid pet22b（＋）. The E.coli BL21 was transformed with the recombinant plasmids and induced to express the target proteins. The expressed proteins were identified by SDS-PAGE and their activity was measured colourimetrically. Results： The products of PCR identified by agrose gel electrophoresis were about 2 kb in length. The relative molecular weights of expressed products were 29 000 and 44 000 respectively, as determined by SDS-PAGE. The specific enzyme activity was increased by 3.7-fold compared to the control. Conclusion： The recombinant plasmid pet22b（＋）-trpBA was constructed successfully. This formed a basis for further construction of the genetic engineering bacteria in high tryptophan production.