A novel method for the production of in vivo-assembled, recombinant Escherichia coli RNA polymerase lacking the alpha C-terminal domain

作者：Twist Kelly Anne; Husnain Seyyed I; Franke Josef D; Jain Deepti; Campbell Elizabeth A; Nickels Bryce E; Thomas Mark S; Darst Seth A^*; Westblade Lars F

来源：Protein Science, 2011, 20(6): 986-995.

DOI：10.1002/pro.622

• RNA polymerase
• alpha-subunit
• macromolecular complex
• purification

The biochemical characterization of the bacterial transcription cycle has been greatly facilitated by the production and characterization of targeted RNA polymerase (RNAP) mutants. Traditionally, RNAP preparations containing mutant subunits have been produced by reconstitution of denatured RNAP subunits, a process that is undesirable for biophysical and structural studies. Although schemes that afford the production of in vivo-assembled, recombinant RNAP containing amino acid substitutions, insertions, or deletions in either the monomeric beta or beta' subunits have been developed, there is no such system for the production of in vivo-assembled, recombinant RNAP with mutations in the homodimeric alpha-subunits. Here, we demonstrate a strategy to generate in vivo-assembled, recombinant RNAP preparations free of the alpha C-terminal domain. Furthermore, we describe a modification of this approach that would permit the purification of in vivo-assembled, recombinant RNAP containing any alpha-subunit variant, including those variants that are lethal. Finally, we propose that these related approaches can be extended to generate in vivo-assembled, recombinant variants of other protein complexes containing homomultimers for biochemical, biophysical, and structural analyses.

• 出版日期2011-6