title: Mutants with higher stability and specific activity from a single thermosensitive variant of T7 RNA polymerase.
abstract: A single strategy to select RNA polymerase from bacteriophage T7 (T7 RNAP) mutants in Escherichia coli with enhanced thermostability or enzymatic activity is described. T7 RNAP has the ability to specifically transcribe genes under control of T7 phage promoter. By using random mutagenesis of the T7 RNAP gene in combination with an appropriate screening at 25 and 42°C, we have generated and selected E.coli clones with temperature-sensitive phenotype in the presence of chloramphenicol. The resistance to chloramphenicol used to select these clones results from expression control of the chloramphenicol acetyl transferase gene by the T7 promoter. In a second phase, and using the thermosensitive T7 RNAP variants as template, a new round of random mutagenesis was performed. Combined to an appropriate screening strategy, 11 mutations (second-site T7 RNAP revertants) that restore the initial resistance to chloramphenicol at 42°C were identified. Nine of these mutations increase the thermal resistance of the wild-type T7 RNA. They include the five mutations previously described using different approaches and four novel mutations. One improves T7 RNA catalytic activity and one has no positive effect on the natural enzyme but increases the activity of some combined mutants. Additive effects of mutations amount to an increase of as much as 10°C in T1/2 compared with the wild-type enzyme and up to a 2-fold activity enhancement.
authors.affiliation: CEA, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, Laboratoire d'Ingénierie des Anticorps pour la Santé, Equipe mixte CEA/BioMérieux, Gif-sur-Yvette F-91191, France.
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Boulain
authors.forename: Janie
authors.initials: J
authors.lastname: Dassa
authors.forename: Laurent
authors.initials: L
authors.lastname: Mesta
authors.forename: Alexandra
authors.initials: A
authors.lastname: Savatier
authors.forename: Narciso
authors.initials: N
authors.lastname: Costa
authors.forename: Bruno H
authors.initials: BH
authors.lastname: Muller
authors.forename: Guillaume
authors.initials: G
authors.lastname: L'hostis
authors.forename: Enrico A
authors.initials: EA
authors.lastname: Stura
authors.forename: Alain
authors.initials: A
authors.lastname: Troesch
authors.forename: Frédéric
authors.initials: F
authors.lastname: Ducancel
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
keywords: T7 RNAP variants; combined mutations; error-prone PCR; suppressor mutations; two-plasmid system