Overproduction and purification of recombinant Bacillus subtilis RNA polymerase.
We describe a vector-based system for the production of recombinant Bacillus subtilis RNA polymerase. The recombinant enzyme is C-terminally tagged with nine consecutive histidine residues resulting in about 90% pure enzyme in a single nickel-affinity purification step. The vectors permitted production of recombinant enzyme lacking an omega subunit or containing either the omega(1) (YkzG) or omega(2) (YloH) subunits. In transcription time-course assays all of the recombinant enzymes exhibited identical activity to native RNAP. The modular assembly of the artificial RNA polymerase operon permits ready mutation of any subunit and incorporation into the recombinant enzyme, which will enable new functional/structural studies with this enzyme.
Yang
Xiao
X
School of Environmental and Life Sciences, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.
Lewis
Peter J
PJ
D001412:Bacillus subtilis / Q000201:enzymology*; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D009876:Operon; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases