Expression of highly toxic genes in E. coli: special strategies and genetic tools.
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.
Saïda
F
F
Center for Molecular Genetics, University of California San Diego, 9500 Gilman Drive, La Jolla CA 92093-0634, USA. fsaida@biomail.ucsd.edu
Uzan
M
M
Odaert
B
B
Bontems
F
F
D001709:Biotechnology / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000633:toxicity*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008957:Models, Genetic; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D029968:Escherichia coli Proteins