利用双启动子表达载体pDH2-YggG-P_(tac)-Era研究E.coli Era和YggG蛋白间的相互关系  

Analysis of the Relationship Between Era and YggG in E.coli by Double-promoter Expression Vector pDH2-YggG-P_(tac)-Era

作　　者：黄勇[1] 张晓楠[1] 王涛[1] 王丽[1] 关路媛[1] 陈南春[1] 陈苏民[1] 

机构地区：[1]中国人民解放军第四军医大学生物化学与分子生物学教研室,西安710032

出　　处：《中国生物工程杂志》2007年第3期12-18,共7页China Biotechnology

摘　　要：yggG是从大肠杆菌全基因组文库中钓取并克隆的Era结合蛋白基因,研究表明该基因可能表达产物YggG294蛋白对宿主菌的生长具有强烈的抑制作用。为了阐明YggG294所引起的细菌生长抑制是否与Era相关,构建可同时可控性表达Era和YggG294蛋白的双启动子表达载体。利用所构建的双启动子表达载体在同一细胞中同时可控性地表达YggG294与Era蛋白。结果显示,在不表达和少量表达YggG294的细菌细胞内,Era的表达量与总蛋白量的比值随着诱导时间增加而增高,而YggG294大量表达的细菌内Era的表达量未明显改变,并且Era的表达量与总蛋白量的比值基本保持不变,但是在全细胞裂解液中并未检测到Era蛋白的降解产物;Era蛋白的预表达对YggG294表达所引起的细菌生长率下降无影响。上述结果说明,YggG294对Era蛋白无水解作用,YggG与Era蛋白间的相互作用与YggG294对细菌生长抑制作用无直接的联系。由此可以推论,Era与YggG294引起的细菌生长抑制无关,YggG294是通过其它途径引起宿主菌生长抑制。yggG, a Era-binding protein gene, was isolated and cloned from the E. coli genomic DNA library. Previous studies indicated that the product of yggG gene, YggG294 （amino acids 1 -294 ）, strongly inhibited the growth of host bacteria and caused the death of bacteria cells. To elucidate whether Era is related to the death of bacterial cells expressed YggG294, A double promoter expression vector that can express YggG294 and Era proteins controllably in cells was constructed. Using this vector to express YggG294 and Era protein in the same E. coli cells, then analyzed the relation between YggG294 and Era. The results showed that the ratio of Era proteins to total proteins increased with the increase of induction time in E. coli cells without YggG294 expression and with little YggG294 expression;the ratio of Era proteins to total proteins seemed to be a constant level in E. coli cells overexpressing YggG294; but we could not detect any Era hydrolyzate in E. coli cells overexpressed YggG294 could not be detected. The results also showed that pre-expression of Era protein did not produce any effect on the growth inhibition of E. coli cells caused by YggG294. These results indicate that YggG294 can not hydrolyze Era protein in E. coli cells, and that YggG-Era interaction is not associated with the death of bacteria expressed YggG294. It is thus reasonable to draw a conclusion that Era is not associated with the growth inhibition of E. coli cells caused by YggG294. YggG294 inhibits the growth of bacteria by other way.

关 键 词：双启动子表达载体 YggG蛋白 ERA蛋白 功能研究 

分 类 号：Q786[生物学—分子生物学]