Rapid purification of His(6)-tagged Bacillus subtilis core RNA polymerase

Protein Expr Purif. 2000 Aug;19(3):350-4. doi: 10.1006/prep.2000.1272.

Abstract

Bacillus subtilis core RNA polymerase, containing a His(6)-fusion to the C-terminus of the beta' subunit, was isolated by Ni-NTA, Superdex 200 gel filtration, and Mono Q anion-exchange chromatography. The purified core enzyme was shown to be free of the major sigma factor(A) and the transcription factors NusA and GreA. The purification procedure can be completed within 1 working day, is scalable, and yields highly purified and active core RNA polymerase.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacillus subtilis / genetics
  • Bacillus subtilis / metabolism*
  • Chromatography
  • Chromatography, Affinity
  • DNA-Directed RNA Polymerases / genetics*
  • DNA-Directed RNA Polymerases / isolation & purification*
  • DNA-Directed RNA Polymerases / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Activation
  • Gene Expression Regulation, Bacterial / physiology
  • Molecular Probes
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / isolation & purification*
  • Recombinant Fusion Proteins / metabolism
  • Transcription Factors / metabolism
  • Transcription, Genetic

Substances

  • Molecular Probes
  • Recombinant Fusion Proteins
  • Transcription Factors
  • beta' subunit of RNA polymerase
  • DNA-Directed RNA Polymerases