New System for Positive Selection of Recombinant Plasmids and Dual Expression in Yeast and Bacteria Based on the Restriction Ribonuclease RegB

• Published in: Biotechnology progress 2003-05, Vol.19 (3), p.727-733
• Main Authors: Saïda, Fakhri, Uzan, Marc, Lallemand, Jean-Yves, Bontems, François
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological and medical sciences; Biotechnology; Cloning, Molecular - methods; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Fungal Proteins - biosynthesis; Fungal Proteins - genetics; Gene Expression Regulation, Bacterial - physiology; Gene Expression Regulation, Fungal - physiology; Plasmids - genetics; Promoter Regions, Genetic; Protein Engineering - methods; Protein Kinases - genetics; Protein Kinases - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Ribosomal Proteins - biosynthesis; Ribosomal Proteins - genetics; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Selection, Genetic
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1021/bp0257224

By coupling the toxic restriction endoribonuclease RegB, 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.