Antigen-binding properties of monoclonal antibodies reactive with human TATA-binding protein and use in immunoaffinity chromatography

• Published in: Protein expression and purification 2004-08, Vol.36 (2), p.186-197
• Main Authors: Thompson, Nancy E, Foley, Katherine M, Burgess, Richard R
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; Chromatography, Affinity - methods; Epitopes - chemistry; Epitopes - genetics; Epitopes - immunology; Escherichia coli - genetics; Gene Expression; HeLa Cells; Humans; Immunoaffinity; mAbs; Monoclonal antibodies; Plasmids; Polyol; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification; TATA-binding protein; TATA-Box Binding Protein - genetics; TATA-Box Binding Protein - immunology; TATA-Box Binding Protein - isolation & purification; TBP; Transcription
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2004.02.020

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.