Premium
Purification of Escherichia coli RNA polymerase using a self‐cleaving elastin‐like polypeptide tag
Author(s) -
Fong Baley A.,
Gillies Alison R.,
Ghazi Iraj,
LeRoy Gary,
Lee Kathleen C.,
Westblade Lars F.,
Wood David W.
Publication year - 2010
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.403
Subject(s) - intein , escherichia coli , rna polymerase , recombinant dna , protein subunit , biochemistry , polymerase , chemistry , t7 rna polymerase , peg ratio , biology , microbiology and biotechnology , rna , enzyme , bacteriophage , gene , finance , rna splicing , economics
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 α subunit was tagged with a self‐cleaving ELP‐intein tag and coexpressed with the β, β′, and ω 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.