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

作者：Yang Xiao; Ma Cong; Lewis Peter^*

来源：Plasmid, 2014, 75: 37-41.

DOI：10.1016/j.plasmid.2014.06.002

• RNA polymerase
• Escherichia coli
• Dual-expression
• Transcription

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%26apos; subunit to facilitate protein purification. Unique restriction sites were introduced into the genes encoding the beta and beta%26apos; 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%26apos; subunit, which is essential for interaction with transcription initiation factor sigma, was generated and exhibited reduced activity compared to native recombinant RNAP.

• 出版日期2014-9