Protein Expression and PuriWcation 36 (2004) 186–197 www.elsevier.com/locate/yprep 1046-5928/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2004.02.020 Antigen-binding properties of monoclonal antibodies reactive with human TATA-binding protein and use in immunoaYnity chromatography Nancy E. Thompson, ¤ Katherine M. Foley, and Richard R. Burgess McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI 53706, USA Received 12 December 2003, and in revised form 10 February 2004 Available online 20 May 2004 Abstract 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 immunoaYnity 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 centri- fugation, the supernatant Xuid was applied to an immunoadsorbent containing mAb 1TBP22. After extensive washing, the TBP was eluted with buVer containing 0.75 M ammonium sulfate and 40% propylene glycol. Human TPB puriWed by the immunoaYnity chro- matography 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.  2004 Elsevier Inc. All rights reserved. Keywords: TATA-binding protein; TBP; ImmunoaYnity; Polyol; Monoclonal antibodies; mAbs; Transcription Initiation of transcription by eukaryotic RNA polymerases requires that a variety of proteins interact in the promoter region of the gene. One protein that is required in most cases for transcription by RNA polymerases I, II, and III (reviewed in [1]) is the TATA- binding protein (TBP). 1 TBP assembles with other proteins to form unique multimeric complexes for each of the three diVerent nuclear RNA polymerases (reviewed in [2,3]). For transcription from RNA poly- merase II (RNAP II) promoters, the complex is TFIID [reviewed in [4,5]], which contains TBP and about 12 TBP-associated factors (TAFs). However, TBP alone can direct basal transcription in vitro from some promoters. TBP has been cloned from a variety of species and consists of two distinct domains. The carboxyl-terminal (C-terminal) domain (about 180 amino acids) is highly conserved among species; however, the amino-terminal (N-terminal) domain varies considerably in length and sequence among species (reviewed in [2]). The conserved C-terminal domain contains the DNA-binding region as well as regions that interact with positive and nega- tive regulatory proteins, including the TAF proteins. The crystal structure of the C-terminal domain bound to DNA [6,7] shows a “saddle-shaped” protein, which ¤ Corresponding author. Fax: 1-608-262-2824. E-mail address: thompson@oncology.wisc.edu (N.E. Thompson). 1 Abbreviations used: mAb(s), monoclonal antibody (antibodies); PR-mAb(s), polyol-responsive mAb(s); TBP, TATA-binding protein; TFIIB, transcription factor IIB; TAFs, TBP-associated factors; RNAP II, RNA polymerase II; N-terminal, amino-terminal; C-terminal, car- boxyl-terminal; NE, nuclear extract; ELISA, enzyme-linked-immuno- sorbent assay; PEI, polyethyleneimine; PBS, phosphate-buVered saline; DEAE, diethylaminoethyl; ECL, enhanced chemiluminescence; Ad- MLP, adenovirus 2 major late promoter; IgH, immunoglobulin heavy chain; BSA, bovine serum albumin; IgG, immunoglobulin G; bp, base pair.