A cross-reactive polyol-responsive monoclonal antibody useful for isolation of core RNA polymerase from many bacterial species.
The use of antibodies for protein purification is a powerful technique but the release of the target protein in its active form is often difficult. So called "polyol-responsive" monoclonal antibodies (PR-MAbs) have a feature that allows elution of the antigen under very gentle conditions, so that even multi-subunit proteins can be released in their active form. In this work a PR-MAb, 8RB13, was isolated that can purify RNA polymerase (RNAP) from many different bacterial species. High specificity towards RNAP with a broad species cross-reactivity was achieved by immunization with RNAP from Escherichia coli and screening with Bacillus subtilis RNA polymerase. The isolated MAb could detect the beta-subunit of RNA polymerase from 10 out of 12 species tested on a Western blot indicating its potential for purification of core RNAP from these organisms. Representatively, four of these species E. coli, B. subtilis, Pseudomonas aeruginosa, and Streptomyces coelicolor were subjected to immunoaffinity purification yielding RNA polymerases that were active in in vitro transcription and seemed to be primarily core polymerase, lacking sigma-subunits.
Bergendahl
Veit
V
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, 1400 University Avenue, Madison, WI 53706, USA. veit@oncology.wisc.edu
Thompson
Nancy E
NE
Foley
Kit M
KM
Olson
Brian M
BM
Burgess
Richard R
RR
D016960:Agrobacterium tumefaciens / Q000201:enzymology / Q000276:immunology; D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D001202:Ascitic Fluid / Q000737:chemistry; D001412:Bacillus subtilis / Q000201:enzymology / Q000276:immunology; D001419:Bacteria / Q000201:enzymology* / Q000276:immunology; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods; D003429:Cross Reactions / Q000276:immunology; D012321:DNA-Directed RNA Polymerases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology / Q000276:immunology; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D011108:Polymers / Q000737:chemistry; D019946:Propylene Glycol / Q000737:chemistry; D011549:Pseudomonas / Q000201:enzymology / Q000276:immunology; D012761:Shigella boydii / Q000201:enzymology / Q000276:immunology; D013302:Streptomyces / Q000201:enzymology / Q000276:immunology
D000911:Antibodies, Monoclonal; D011108:Polymers; C024617:polyol; D019946:Propylene Glycol; C108570:beta' subunit of RNA polymerase; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D000645:Ammonium Sulfate