大肠杆菌trpBA基因的克隆表达

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。

Cloning and Expression of trpBA Genes from E. coli

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.