Publication:
Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis
Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis
No Thumbnail Available
Date
2017
Authors
Herrera-Asmat O.
Lubkowska L.
Kashlev M.
Bustamante C.J.
Guerra D.G.
Kireeva M.L.
Journal Title
Journal ISSN
Volume Title
Publisher
Elsevier B.V.
Research Projects
Organizational Units
Journal Issue
Abstract
Recent publications have shown that active RNA polymerase (RNAP) from Mycobacterium tuberculosis (MtbRNAP) can be produced by expressing all four subunits in a single recombinant Escherichia coli strain [1–3]. By reducing the number of plasmids and changing the codon usage of the Mtb genes in the co-expression system published by Banerjee et al. [1], we present a simplified, detailed and reproducible protocol for the purification of recombinant MtbRNAP containing the ω subunit. Moreover, we describe the formation of ternary elongation complexes (TECs) with a short fluorescence-labeled RNA primer and DNA oligonucleotides, suitable for transcription elongation studies.
Description
Keywords
recombinant protein,
bacterial protein,
DNA directed RNA polymerase,
holoenzyme,
biosynthesis,
chemistry,
enzymology,
Escherichia coli,
genetic transcription