Isolation and characterization of a polyol-responsive monoclonal antibody useful for gentle purification of Escherichia coli RNA polymerase.
A modified enzyme-linked immunosorbent assay (ELISA) was used to screen monoclonal antibodies (MAbs) that react with Escherichia coli RNA polymerase for the ability to release the RNA polymerase in the presence of a low molecular weight polyhydroxylated compound (polyol) and a non-chaotropic salt. This assay, termed the ELISA-elution assay, identified 19 presumptive "polyol-responsive" MAbs out of a total of 218 antigen-specific MAbs screened. One of these MAbs, designated NT73, was examined in detail for the ability to release the antigen in response to various combinations of polyol and salt. Using NT73 conjugated to Sepharose, highly active RNA polymerase could be prepared rapidly by a single immunoaffinity chromatography step, replacing two lengthy chromatographic steps in our conventional purification procedure. Because NT73 reacts with the beta' subunit of RNA polymerase, a mixture of the core polymerase and holoenzyme was recovered from the immunoaffinity column. The holoenzyme (E sigma 70) could be separated from the core polymerase by subsequent chromatography on a Mono Q column. This demonstrates that polyol-responsive MAbs can be easily identified and characterized by the ELISA-elution assay. The use of polyol-responsive MAbs provides a means of adapting immunoaffinity chromatography to the purification of labile proteins.
Thompson
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NE
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison 53706.
Hager
D A
DA
Burgess
R R
RR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000187:drug effects; D002846:Chromatography, Affinity; D012321:DNA-Directed RNA Polymerases / Q000276:immunology / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology*; D005260:Female; D006825:Hybridomas / Q000276:immunology; D007158:Immunologic Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D011108:Polymers / Q000494:pharmacology*; D012965:Sodium Chloride / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D011108:Polymers; C024617:polyol; D012965:Sodium Chloride; D012321:DNA-Directed RNA Polymerases