大肠杆菌邻氨基苯甲酸合成酶编码基因trpED的克隆与表达  

The Clone and Expression of the Anthranilate Synthetase Coding Gene in Escherichia Coli

作　　者：李剑欣[1] 郭长江[2] 刘云[1] 林维平[3] 张绪梅[2] 徐琪寿[1] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850 [2]军事医学科学院食品与环境卫生研究所,天津300050 [3]南昌大学生命科学学院,江西南昌330000

出　　处：《生物技术通讯》2007年第2期183-185,共3页Letters in Biotechnology

基　　金：国家自然科学基金项目(30300010);天津市科技攻关项目(033182711)

摘　　要：目的:通过基因重组的方法获得在体外具有邻氨基苯甲酸合成酶活性的基因trpED,为进一步解除反馈抑制的基因改造寻找靶标。方法:分别构建重组质粒pBV220-trpE和pBV220-trpD;由于trpE和trpD存在翻译偶联现象,将其自大肠杆菌基因组中经PCR扩增得到,构建新的表达质粒pBV220-trpED。结果:SDS-PAGE显示,包含质粒pBV220-trpED的重组菌在相对分子质量约53000(trpE基因表达)和56000(trpD基因表达)处的蛋白表达量明显增多,且邻氨基苯甲酸合成酶活性是对照的3倍。结论:翻译偶联的存在直接影响蛋白活性的发挥;重组质粒pBV220-trpED的获得为进一步的基因改造,以提高色氨酸产量奠定了研究基础。Objective： To get the most active anthranilate synthetase coding gene in vitro, and to build the fundation for the further releasing feed-back inhibition. Methods： Because of the overlapping gene between trpE and trpD, the trpED was cloned integralhy to produce a new high-expression vector pBV220-trpED. Results; The SDS-PAGE indicated that the protein expression of the recombined bacterium had an obvious increase in aim sites, and the enzyme assays showed the recombined one has three times activity of the antitheses. Conclusion： The overlapping phenomenon in Escherichia coli directly influenced the protein activity; and all of these production and data will be elements of the following work to improve the output of tryptophan.

关 键 词：trpED基因 邻氨基苯甲酸合成酶 色氨酸 生物合成 

分 类 号：Q78[生物学—分子生物学]