State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, P.R. China.
BMC Biotechnol. 2013 Mar 19;13:25. doi: 10.1186/1472-6750-13-25.
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.
By using GFP as reporter, PfhuA with the highest relative fluorescence units, but leaky expression, was screened from 23 iron-regulated promoter candidates. PfhuA was repressed by ferric uptake regulator (Fur)-Fe2+ complex binding to Fur box locating at the promoter sequence. Otherwise, PfhuA was activated without Fur-Fe2+ binding in the absence of iron. In order to improve the tightness of PfhuA 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 PfhuA, the induction ratio of modified PfhuA (named PfhuA1) was improved from 3 to 101. Under the control of PfhuA1, 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.
The features of easy control, tight regulation and relatively high efficiency were combined in the newly engineered PfhuA1. 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.
人们对严格调控的原核表达系统的需求持续增长，该系统允许在大肠杆菌中功能性合成毒性蛋白，从而应用于生物科学或生物技术领域。然而，目前大多数启动子选项要么受到严格抑制，蛋白产量水平较低，要么产生大量蛋白，但缺乏在没有诱导剂时避免由漏表达引发的突变所必需的抑制作用。本研究旨在基于铁摄取系统的原理，在大肠杆菌中开发一种严格调控、相对高效的表达载体。
通过使用 GFP 作为报告基因，从 23 个铁调控启动子候选物中筛选出相对荧光单位最高、但存在漏表达的 PfhuA。PfhuA 受到 Fur-Fe2+ 复合物与位于启动子序列中的 Fur 盒结合的抑制。否则，在没有铁的情况下，PfhuA 在没有 Fur-Fe2+ 结合时被激活。为了提高 PfhuA 调控的严密性，以用于毒性基因表达，启动子序列中的 Fur 盒和 fur 表达通过五种不同的方法进行了优化。最终，通过用大肠杆菌共识 Fur 盒替代 PfhuA 的原始 Fur 盒，修饰后的 PfhuA（命名为 PfhuA1）的诱导比从 3 提高到 101。在 PfhuA1 的控制下，强毒性基因 E 在高、中、低拷贝数载体中成功表达，在诱导前没有大肠杆菌死亡，其他两种毒性蛋白 Gef 和 MazF 也被功能性合成。
新设计的 PfhuA1 结合了易于控制、严格调控和相对高效的特点。在该启动子的控制下，毒性基因 E、gef 和 mazF 在大肠杆菌中被铁螯合剂诱导，以一种严格可控的方式实现功能性表达。本研究提供了一种严格调控的表达系统，可用于稳定克隆和毒性蛋白的功能性合成，从而研究其功能、在生物安全系统中实现细菌程序性细胞死亡以及开发细菌载体疫苗。
