A vector system that allows simple generation of mutant Escherichia coli RNA polymerase

摘要

We describe a dual vector-based system for overproduction of recombinant Escherichia coli RNA polymerase (RNAP). A cleavable deca-histidine tag (His10) was incorporated into the C-terminus of the beta' subunit to facilitate protein purification. Unique restriction sites were introduced into the genes encoding the beta and beta' subunits (rpoB and rpoC, respectively), facilitating mutation of functionally significant subunit fragments through insertion of modified PCR fragments into the appropriate vector. RNAP with an R275A substitution in the beta' subunit, which is essential for interaction with transcription initiation factor sigma, was generated and exhibited reduced activity compared to native recombinant RNAP. Crown Copyright (C) 2014 Published by Elsevier Inc. All rights reserved.