title: The epitope for the polyol-responsive monoclonal antibody 8RB13 is in the flap-domain of the beta-subunit of bacterial RNA polymerase and can be used as an epitope tag for immunoaffinity chromatography.
abstract: Polyol-responsive monoclonal antibodies (PR-mAbs) are useful for the purification of proteins in an easy, one step immunoaffinity step. These antibodies allow for gentle purification of proteins and protein complexes using a combination of a low molecular weight polyhydroxylated compound (polyol) and a nonchaotrophic salt in the eluting buffer. mAb 8RB13 has been characterized as one of these PR-mAbs and has been used to purify RNA polymerase from five species of bacteria. Here the epitope for 8RB13 has been identified as PEEKLLRAIFGEKAS, a sequence that is highly conserved in the β-subunit of bacterial RNA polymerase. This sequence is located in the "beta-flap" domain of RNA polymerase (and essentially comprises the "flap-tip helix"), an important binding site for sigma70. This location explains why only the core RNAP is purified using this mAb. This amino acid sequence has been developed into an epitope tag that can be used to purify a target protein from either bacterial or eukaryotic cells when genetically fused to a protein of interest.
authors.affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI 53706, United States.
authors.forename: Elizabeth S
authors.initials: ES
authors.lastname: Stalder
authors.forename: Lauren H
authors.initials: LH
authors.lastname: Nagy
authors.forename: Pilar
authors.initials: P
authors.lastname: Batalla
authors.forename: Terrance M
authors.initials: TM
authors.lastname: Arthur
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Thompson
authors.forename: Richard R
authors.initials: RR
authors.lastname: Burgess
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D012321:DNA-Directed RNA Polymerases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D018604:Epitope Mapping; D000939:Epitopes; D004926:Escherichia coli; D049452:Green Fluorescent Proteins / Q000302:isolation & purification / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007163:Immunosorbent Techniques; D008164:Luminescent Proteins / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D011108:Polymers; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000939:Epitopes; D008164:Luminescent Proteins; D011108:Polymers; D011993:Recombinant Fusion Proteins; C024617:polyol; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit