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.
Submitter: Shu-Ching Ou
Submission Date: Dec. 7, 2018, 1:10 p.m.
|Number of data points||171|
|Proteins||T7 RNA polymerase|
|Assays/Quantities/Protocols||Experimental Assay: Specific Activity ; Experimental Assay: Half-Life Temperature After 10-min Pre-Incubation (Ti_1/2) ; Experimental Assay: Specific Activity at 37 °C ; Derived Quantity: Relative Activity ; Derived Quantity: Difference in Half-Life Temperature (ΔTi_1/2)|
|Libraries||Variants for T7-RNAp|