文献类型：期刊文献

英文题名：Development of a Fur-dependent and tightly regulated expression system in Escherichia coli for toxic protein synthesis

作者：Guan, Lingyu[1];Liu, Qin[1];Li, Chao[1];Zhang, Yuanxing[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2013

卷号：13

外文期刊名：BMC BIOTECHNOLOGY

收录：;EI(收录号：20131616215128);WOS:【SCI-EXPANDED(收录号:WOS:000317392900001)】；

基金：This work was supported by the joint project, National Natural Science Foundation of China- Austrian Science Fund (30811130545).

语种：英文

外文关键词：Proteins - Gene expression regulation - Iron compounds - Vectors - Cell death - Biosynthesis - Gene expression

摘要：Background: There is a continuous demanding for tightly regulated prokaryotic expression systems, which allow functional synthesis of toxic proteins in Escherichia coli for bioscience or biotechnology application. However, most of the current promoter options either are tightly repressed only with low protein production levels, or produce substantial protein but lacking of the necessary repression to avoid mutations initiated by leaky expression in the absence of inducer. The aim of this study was to develop a tightly regulated, relatively high-efficient expression vector in E. coli based on the principle of iron uptake system. Results: By using GFP as reporter, P-fhuA with the highest relative fluorescence units, but leaky expression, was screened from 23 iron-regulated promoter candidates. P-fhuA was repressed by ferric uptake regulator (Fur)-Fe2+ complex binding to Fur box locating at the promoter sequence. Otherwise, P-fhuA was activated without Fur-Fe2+ binding in the absence of iron. In order to improve the tightness of P-fhuA regulation for toxic gene expression, Fur box in promoter sequence and fur expression were refined through five different approaches. Eventually, through substituting E. coli consensus Fur box for original one of P-fhuA, the induction ratio of modified P-fhuA (named P-fhuA1) was improved from 3 to 101. Under the control of P-fhuA1, strong toxic gene E was successfully expressed in high, middle, low copy-number vectors, and other two toxic proteins, Gef and MazF were functionally synthesized without E. coli death before induction. Conclusions: The features of easy control, tight regulation and relatively high efficiency were combined in the newly engineered P-fhuA1. Under this promoter, the toxic genes E, gef and mazF were functionally expressed in E. coli induced by iron chelator in a tightly controllable way. This study provides a tightly regulated expression system that might enable the stable cloning, and functional synthesis of toxic proteins for their function study, bacterial programmed cell death in biological containment system and bacterial vector vaccine development.