Antigen-binding properties of monoclonal antibodies reactive with human TATA-binding protein and use in immunoaffinity chromatography.
The TATA-binding protein (TBP) plays a central role in the assembly of most eukaryotic transcription initiation complexes. We have characterized 3 monoclonal antibodies (mAbs) that react in the far amino-terminal (N-terminal) domain of the human TBP molecule (residues 1-99). One of these mAbs (designated 1TBP22) is a polyol-responsive monoclonal antibody (PR-mAb) and was adapted to an immunoaffinity chromatography procedure for purifying bacterially expressed, recombinant human TBP. The epitope for mAb 1TBP22 maps to residues 55-99, which includes the polyglutamine region. However, mAb 1TBP22 does not react with poly-l-glutamine. Human TBP, contained on the pET11a plasmid, was expressed in Escherichia coli Rosetta (DE3)pLysS. The cell lysate from 330 ml of induced culture was treated with polyethyleneimine (PEI) at 0.5 M NaCl to precipitate the nucleic acids. After centrifugation, the supernatant fluid was applied to an immunoadsorbent containing mAb 1TBP22. After extensive washing, the TBP was eluted with buffer containing 0.75 M ammonium sulfate and 40% propylene glycol. Human TPB purified by the immunoaffinity chromatography method was found to be active in gel-shift assays and transcription assays. Preliminary data indicate that this mAb might be useful for purifying protein complexes containing TBP from HeLa cell extracts.
Thompson
Nancy E
NE
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI 53706, USA. thompson@oncology.wisc.edu
Foley
Katherine M
KM
Burgess
Richard R
RR
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods*; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D035181:TATA-Box Binding Protein / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D035181:TATA-Box Binding Protein