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New System for Positive Selection of Recombinant Plasmids and Dual Expression in Yeast and Bacteria Based on the Restriction Ribonuclease Reg B
Author(s) -
Saïda Fakhri,
Uzan Marc,
Lallemand JeanYves,
Bontems François
Publication year - 2003
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp0257224
Subject(s) - plasmid , biology , escherichia coli , multiple cloning site , cloning vector , cloning (programming) , saccharomyces cerevisiae , expression vector , recombinant dna , yeast , shuttle vector , bacteria , vector (molecular biology) , genetics , dna , gene , computer science , programming language
By coupling the toxic restriction endoribonuclease Reg B, from the bacteriophage T4, to the prokaryotic T7 and the eukaryotic GAL1 promoters, we constructed a two‐function plasmid called pTOXR‐1. This plasmid is a zero‐background cloning vector. It allows an efficient positive selection of recombinant plasmids without the need to completely digest, dephosphorylate, or purify the vector prior to the ligation step. The pTOXR‐1 positive selection system requires no special Escherichia coli strains, no special culture media, and no addition of inducer to the selective plates. In addition, since this vector carries all signals required for both prokaryotic and eukaryotic expression, it allows the overproduction of heterologous proteins, fused to a polyhistidine tag, in the bacterium E. coli and in the yeast Saccharomyces cerevisiae from a single plasmid. Hence, this vector may be a useful time‐saving tool for one‐step cloning and versatile protein expression in bacteria and yeast.