大肠埃希菌trp operon的克隆与表达

Cloning and Expression of trp Operon in Escherichia coli

色氨酸操纵子所表达酶的高效表达和酶活性的提高,从而构建高产色氨酸菌株。利用PCR的方法从大肠埃希菌基因组中直接克隆色氨酸操纵子,并将其连接到原核表达载体pBV220中,得到重组质粒pBV220-trp operon,转化大肠埃希菌DH5α,温度诱导重组蛋白表达,表达产物经SDS-PAGE分析并用比色法测定其活性。通过凝胶电泳观察PCR扩增产物大小约为7 kb,SDS-PAGE鉴定目的蛋白得到了高效表达,邻氨基苯甲酸合成酶和色氨酸合成酶的活性分别比对照提高了3.4倍和2.5倍。成功构建了重组质粒pBV220-trp operon,邻氨基苯甲酸合成酶和色氨酸合成酶的表达量和表达活性在大肠埃希菌中得到了提高,为高产色氨酸基因工程菌的构建奠定基础。

Using PCR method tryptophane operon was directly cloned from E. coli genome and then link it into prokaryotic expression vector pBV220 and obtained recombinant pBV220-trp operon and transformed E. coli DH5α to increase the high efficient expressive enzyme of tryptophane operon and enzyme activity thus to construct a tryptophane high - yielding strain. The recombinant protein was inductively expressed with temperature, and the expression target was analyzed with SDS-PAGE and tested its activity with colorimetry. The product of PCR observed by agarose gel e- leetrophoresis was about 7 kb, the purpose protein identified with SDS-PAGE had gained high efficient expression, and the activity of anthranilate synthetase and tryptophane synthetase enhanced 3.4 times and 2.5 time respectively as compared with the eontrol. Therefore, the reeombinant plasmid pBV220-trp operon was constructed successfully. The increment of the expression volume and activity of anthranilate synthetase and tryptophane synthetase in E. coli had laid the foundation of the construetion of the genetically engineered tryptophane high-yielding strain.