大肠杆菌色氨酸操纵子基因的克隆与表达  

Cloning and Expression of Trp Operon Gene of E.coli

作　　者：林维平[1] 郭长江[2] 张绪梅[2] 徐琪寿[3] 

机构地区：[1]潍坊医学院基础医学部,潍坊261042 [2]军事医学科学院食品与环境卫生研究所,天津300050 [3]军事医学科学院放射医学研究所,北京100850

出　　处：《中国生物制品学杂志》2008年第1期30-32,共3页Chinese Journal of Biologicals

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

摘　　要：目的克隆并表达大肠杆菌色氨酸操纵子基因,提高其酶活性。方法利用PCR方法,从大肠杆菌31884基因组中直接克隆出色氨酸操纵子,并将其连接到原核表达载体pET-22b(+)中,构建重组质粒pET-22b(+)-Trpoperon,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,并测定酶活性。结果凝胶电泳可见PCR扩增产物大小约为7000bp,SDS-PAGE鉴定目的蛋白分别得到了表达,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2.7和3.2倍。结论已成功构建了重组表达质粒pET-22b(+)-Trpoperon,邻氨基苯甲酸合成酶和色氨酸合成酶的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了基础。Objective To clone and express the Trp operon gene of E. coli and increase the enzyme activity of expressed product. Methods Clone Trp operon directly from the genome of E. coli 31884 by PCR and insert into prokaryotic expression vector pET-22b（ ＋ ）. Transform the constructed recombinant plasmid pET-22b（ ＋ ）-Trp operon to E. coli BL21 for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and determined for enzyme activity. Results Agarose gel electrophoresis proved that the length of PCR product was about 7 000 bp. SDS-PAGE showed that the target protein was highly expressed. Compared with those of control,the anthranilate synthase and tryptophan synthase activities of expressed product increased by 2.7 and 3.2 folds respectively. Conclusion Recombinant plasmid pET-22（ b）-Trp operon was successhtUy constructed, and both the anthranilate synthase and tryptophan synthase activities of expressed product increased. It laid a foundation of construction of recombinant E. coli strain for production of tryptophan in a large scale.

关 键 词：色氨酸操纵子 克隆 表达 酶活性 

分 类 号：Q786[生物学—分子生物学]