Purification of an active TATA-binding protein-containing factor using a monoclonal antibody that recognizes the human TATA-binding protein.
The human TATA-binding protein was expressed in Escherichia coli as a fusion with an N-terminal hexahistidine sequence, partially purified, and used to raise monoclonal antibodies. More than 50 hybridoma clones producing antibodies that reacted in immunoblot assays with HeLa cell TATA-binding protein and its bacterially synthesized derivative were identified. All antibodies examined recognized epitopes within the N-terminal 159 amino acids of the human TATA-binding protein. Further characterization of one monoclonal antibody, MTBP-6, established that it immunoprecipitates both native HeLa cell TATA-binding protein and TATA-binding protein extracted from cells in the presence of 0.5% SDS. Antibody MTBP-6 immunoprecipitates of native, human cell TATA-binding protein contained the TATA-binding protein and additional polypeptides. Immunoprecipitation of both the TATA-binding protein and several additional polypeptides was specifically blocked by bacterially synthesized, hexahistidine-tagged TATA-binding protein, suggesting that MTBP-6 can efficiently recognize the TATA-binding protein in TFIID and other complexes. Consistent with this conclusion, immunoaffinity chromatography on antibody MTBP-6 permitted purification, in active form, of a TATA-binding protein-containing factor required for transcription by RNA polymerase III. These properties suggest that MTBP-6 will be a useful reagent for the purification and characterization of the multiple TBP-containing complexes present in human cells.
Chatterjee
P K
PK
Department of Molecular Biology, Princeton University, New Jersey 08544.
Pruzan
R
R
Flint
S J
SJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D002460:Cell Line; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D005260:Female; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012377:Rodentia; D013045:Species Specificity; D016385:TATA Box; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors