Purification of Escherichia coli RNA polymerase using a self-cleaving elastin-like polypeptide tag Academic Article uri icon

Overview

MeSH Major

  • Chemical Precipitation
  • DNA-Directed RNA Polymerases
  • Elastin
  • Escherichia coli
  • Escherichia coli Proteins

abstract

  • A self-cleaving elastin-like polypeptide (ELP) tag was used to purify the multisubunit Escherichia coli RNA polymerase (RNAP) via a simple, nonchromatographic method. To accomplish this, the RNAP alpha subunit was tagged with a self-cleaving ELP-intein tag and coexpressed with the beta, beta', and omega subunits. The assembled RNAP was purified with its associated subunits, and was active and acquired at reasonable yield and purity. To remove residual polynucleotides bound to the purified RNAP, two polymer precipitation methods were investigated: polyethyleneimine (PEI) and polyethylene (PEG) precipitation. The PEG procedure was shown to enhance purity and was compatible with downstream ELP-intein purification. Thus, this simple ELP-based method should be applicable for the nonchromatographic purification of other recombinant, in vivo-assembled multisubunit complexes in a single step. Further, the simplicity and low cost of this method will likely facilitate scale up for large-scale production of additional multimeric protein targets. Finally, this technique may have utility in isolating protein interaction partners that associate with a given target.

publication date

  • June 2010

Research

keywords

  • Academic Article

Identity

Language

  • eng

PubMed Central ID

  • PMC2895248

Digital Object Identifier (DOI)

  • 10.1002/pro.403

PubMed ID

  • 20512976

Additional Document Info

start page

  • 1243

end page

  • 52

volume

  • 19

number

  • 6