Identification, production, and use of polyol-responsive monoclonal antibodies for immunoaffinity chromatography.

Immunoaffinity chromatography is a powerful tool for purification of proteins and protein complexes. The availability of monoclonal antibodies (mAbs) has revolutionized the field of immunoaffinity chromatography by providing a continuous supply of highly uniform antibody. Before the availability of mAbs, the recovery of the target protein from immobilized polyclonal antibodies usually required very harsh, often denaturing conditions. Although harsh conditions are often still used to disrupt the antigen-antibody interaction when using a mAb, various methods have been developed to exploit the uniformity of the antigen-antibody reaction in order to identify agents or conditions that gently disrupt this interaction and thus result in higher recovery of active protein from immunoaffinity chromatography. We discuss here the use of a specific type of monoclonal antibody that we have designated "polyol-responsive monoclonal antibodies" (PR-mAbs). These are naturally occurring mAbs that have high affinity for the antigen under binding conditions, but have low affinity in the presence of a combination of low molecular weight hydroxylated compounds (polyols) and nonchaotropic salts. Therefore, these PR-mAbs can be used for gentle immunoaffinity chromatography. PR-mAbs can be easily identified and adapted to a powerful protein purification method for a target protein.

Minimal promoter systems reveal the importance of conserved residues in the B-finger of human transcription factor IIB.

The "B-finger" of transcription factor IIB (TFIIB) is highly conserved and believed to play a role in the initiation process. We performed alanine substitutions across the B-finger of human TFIIB, made change-of-charge mutations in selected residues, and substituted the B-finger sequence from other organisms. Mutant proteins were examined in two minimal promoter systems (containing only RNA polymerase II, TATA-binding protein, and TFIIB) and in a complex system, using TFIIB-immunodepleted HeLa cell nuclear extract (NE). Mutations in conserved residues located on the sides of the B-finger had the greatest effect on activity in both minimal promoter systems, with mutations in residues Glu-51 and Arg-66 eliminating activity. The double change-of-charge mutant (E51R:R66E) did not show activity in either minimal promoter system. Mutations in the nonconserved residues at the tip of the B-finger did not significantly affect activity. However, all of the mutations in the B-finger showed at least 25% activity in the HeLa cell NE. Chimeric proteins, containing B-finger sequences from species with conserved residues on the side of the B-finger, showed wild-type activity in a minimal promoter system and in the HeLa cell NE. However, chimeric proteins whose sequence showed divergence on the sides of the B-finger had reduced activity. Transcription factor IIF (TFIIF) partially restored activity of the inactive mutants in the minimal promoter system, suggesting that TFIIF in HeLa cell NE helps to rescue the inactive mutations by interacting with either the B-finger or another component of the initiation complex that is influenced by the B-finger.

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.