Purification of Escherichia coli RNA polymerase using a self-cleaving elastin-like polypeptide tag.
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.
Department of Chemical Engineering, Princeton University, Princeton, New Jersey 08544, USA.
Baley A
BA
Fong
Alison R
AR
Gillies
Iraj
I
Ghazi
Gary
G
LeRoy
Kathleen C
KC
Lee
Lars F
LF
Westblade
David W
DW
Wood
D011232:Chemical Precipitation; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification* / Q000378:metabolism; D004549:Elastin / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D011094:Polyethyleneimine / Q000737:chemistry
D029968:Escherichia coli Proteins; D011092:Polyethylene Glycols; D011094:Polyethyleneimine; D004549:Elastin; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit