Purification of an active tata-binding protein-containing factor using a monoclonal antibody that recognizes the human tata-binding protein

Pradeep K. Chatterjee, Ronald Pruzan, Jane Flint

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

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 TATAbinding protein and several additional polypeptides was specifically blocked by bacterially synthesized, hexahistidine-tagged TATA-binding protein, suggesting that MTBP-6 can efficiently recognize the TATAbinding 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.

Original languageEnglish (US)
Pages (from-to)445-455
Number of pages11
JournalProtein Expression and Purification
Volume4
Issue number5
DOIs
StatePublished - Jan 1 1993

All Science Journal Classification (ASJC) codes

  • Biotechnology

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