Expression of Highly Toxic Genes in E. coli: Special Strategies and Genetic Tools

Abstract

Escherichia coli (E. coli) remains the most efficient widely-used host for recombinant protein production. Well-known genetics, high transformation efficiency, cultivation simplicity, rapidity and inexpensiveness are the main factors that contribute to the selection of this host. With the advent of the post-genomic era has come the need to express in this bacterium a growing number of genes originating from different organisms. Unfortunately, many of these genes severely interfere with the survival of E. coli cells. They lead to bacteria death or cause significant defects in bacteria growth that dramatically decrease expression capabilities. In this paper, we review special strategies and genetics tools successfully used to express, in E. coli, highly toxic genes. Suppression of basal expression from leaky inducible promoters, suppression of read-through transcription from cryptic promoters, tight control of plasmids copy numbers and proteins production as inactive (but reversible) forms are among the solutions presented and discussed. Special expression vectors and modified E. coli strains are listed and their effectiveness illustrated with key examples, some of which are related to our study of the highly toxic phage T4 restriction endoribonuclease RegB. We mainly selected those strategies and tools that permit E. coli normal growth until the very moment of highly toxic gene induction. Expression then occurs efficiently before cells die. Because they do not target a particular toxic effect, these strategies and tools can be used to express a wide variety of highly toxic genes.

High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

“…In the growth phase, the recombinant genes are kept under repressive conditions to prevent their protein expression from interfering with normal growth. Leaky expression under repressive conditions kills host bacteria when the recombinant gene products are highly toxic (39). Since stronger promoters such as P T7 often exhibit greater leaky expression, "high yield" and "zero leakage" have been recognized as incompatible.…”

Strategies for production of active eukaryotic proteins in bacterial expression system

“…E. coli BL21 is an example of the most common host and it has been proven outstanding in application for standard recombinant expression. It can grow efficiently in minimal media as nonpathogenic bacterium that cannot survive to cause diseases in host tissues [9,11,15,16] .…”

“…The increase of expression and activity of lower temperatures growth is associated with increased expression of chaperones in E. coli. Therefore, growth at a temperature range of (15)(16)(17)(18)(19)(20)(21)(22)(23) 曟, could also lead to a significant reduction of expressed protein degradation [4,18,19] .…”

Tight Repression Of Elastase Strain K Overexpression By Pt7 (A1/04/03) Shuttle Expression System

“…While increasing the amount of lactose (>0.05 %), the protein expression remained high with density of saturated culture and viability had decreased substantially. In addition, cells induced using very low IPTG have more metabolic control over the toxic effect than those induced with standard IPTG concentrations [16]. According to Ramirez et al (1994) [17], low IPTG concentrations may result in low recombinant protein yields, whereas expensive IPTG added in excess can result in an important economic loss or in toxic effects, including reduced cell growth and recombinant protein concentration.…”