Weak protein-protein interactions revealed by immiscible filtration assisted by surface tension.

Biological mechanisms are often mediated by transient interactions between multiple proteins. The isolation of intact protein complexes is essential to understanding biochemical processes and an important prerequisite for identifying new drug targets and biomarkers. However, low-affinity interactions are often difficult to detect. Here, we use a newly described method called immiscible filtration assisted by surface tension (IFAST) to isolate proteins under defined binding conditions. This method, which gives a near-instantaneous isolation, enables significantly higher recovery of transient complexes compared to current wash-based protocols, which require reequilibration at each of several wash steps, resulting in protein loss. The method moves proteins, or protein complexes, captured on a solid phase through one or more immiscible-phase barriers that efficiently exclude the passage of nonspecific material in a single operation. We use a previously described polyol-responsive monoclonal antibody to investigate the potential of this new method to study protein binding. In addition, difficult-to-isolate complexes involving the biologically and clinically important Wnt signaling pathway were isolated. We anticipate that this simple, rapid method to isolate intact, transient complexes will enable the discoveries of new signaling pathways, biomarkers, and drug targets.

Production and characterization of monoclonal antibodies to estrogen-related receptor alpha (ERRα) and use in immunoaffinity chromatography.

Estrogen-related receptor alpha (ERRα) is an orphan nuclear receptor whose elevated expression is thought to contribute to breast, colon, and ovarian cancers. In order to investigate the role of ERRα in human disease, there is a need for immunological reagents suitable for detection and purification of ERRα. We expressed recombinant human ERRα in Escherichia coli, purified the protein, and used it to generate monoclonal antibodies (mAbs) to ERRα. Nine high-affinity mAbs were chosen for their abilities to detect overexpressed ERRα in enzyme-linked immunosorbent assays (ELISAs) and Western blots, after which isotyping and preliminary epitope mapping was performed. The mAbs were all IgG subtypes and reacted with several different regions of full-length ERRα. A majority of the mAbs were found to be useful for immunoprecipitation of ERRα, and several could detect DNA-bound ERRα in electrophoretic mobility supershift assays (EMSAs) and chromatin immunoprecipitation (ChIP). The suitability of mAbs to detect ERRα in immunofluorescence assays was assessed. One mAb in particular, 2ERR10, could specifically detect endogenous ERRα in mammary carcinoma cells. Finally, we performed assays to screen for mAbs that gently release ERRα in the presence of a low-molecular-weight polyhydroxylated compound (polyol) and nonchaotropic salt. Using gentle immunoaffinity chromatography, we were able to isolate ERRα from mammalian cells by eluting with a polyol-salt solution. Our characterization studies show that these monoclonal antibodies perform well in a variety of biochemical assays. We anticipate that these novel reagents will prove useful for the detection and purification of ERRα in research and clinical applications.

The epitope for the polyol-responsive monoclonal antibody 8RB13 is in the flap-domain of the beta-subunit of bacterial RNA polymerase and can be used as an epitope tag for immunoaffinity chromatography.

Polyol-responsive monoclonal antibodies (PR-mAbs) are useful for the purification of proteins in an easy, one step immunoaffinity step. These antibodies allow for gentle purification of proteins and protein complexes using a combination of a low molecular weight polyhydroxylated compound (polyol) and a nonchaotrophic salt in the eluting buffer. mAb 8RB13 has been characterized as one of these PR-mAbs and has been used to purify RNA polymerase from five species of bacteria. Here the epitope for 8RB13 has been identified as PEEKLLRAIFGEKAS, a sequence that is highly conserved in the ß-subunit of bacterial RNA polymerase. This sequence is located in the "beta-flap" domain of RNA polymerase (and essentially comprises the "flap-tip helix"), an important binding site for sigma70. This location explains why only the core RNAP is purified using this mAb. This amino acid sequence has been developed into an epitope tag that can be used to purify a target protein from either bacterial or eukaryotic cells when genetically fused to a protein of interest.

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.

LRET-based HTS of a small-compound library for inhibitors of bacterial RNA polymerase.

Resistance mechanisms against whole classes of antibiotics are currently developing faster than research generates new structurally different biologically active agents. The demand for new antimicrobial drugs has not been matched by the speed of discovery. The interface between sigma and core of bacterial RNA polymerase offers an attractive target for drug discovery, and we have previously described the development of a very robust high-throughput assay for this target based on luminescence resonance energy transfer. Here we describe a semiautomated screen of a commercially available library (Chembridge, San Diego, CA) that led to the identification of four small molecules, two of which have activity in preventing in vitro transcription and growth of Escherichia coli.

Immunoaffinity purification and characterization of RNA polymerase from Shewanella oneidensis.

Shewanella oneidensis is of particular interest for research because of its unique ability to use a variety of metals as final respiratory electron acceptors and reduce them into insoluble oxides. A collection of monoclonal antibodies (mAbs) that were prepared towards Escherichia coli RNA polymerase (RNAP) was tested for reactivity with proteins extracted from S. oneidensis. Two polyol-responsive monoclonal antibodies (PR-mAbs) were used to purify RNA polymerase from S. oneidensis using immunoaffinity purification techniques. A collection of mAbs towards E. coli sigma subunits was also examined for cross-reactivity with S. oneidensis proteins. Reactions were identified with mAbs to E. coli sigma(70) and sigma(54). These mAbs will be useful tools for immunoaffinity purifying and studying the transcriptional machinery of S. oneidensis.

Expression and purification of a single-chain variable fragment antibody derived from a polyol-responsive monoclonal antibody.

A previously described polyol-responsive monoclonal antibody (PR-mAb) was converted to a single-chain variable fragment (scFv). This antibody, PR-mAb NT73, reacts with the beta' subunit of Escherichia coli RNA polymerase and has been used for the immunoaffinity purification of polymerase. mRNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were used as the template for cDNA synthesis. The sequences were joined by the addition of a "linker" sequence and then cloned into several expression vectors. A variety of expression plasmids and E. coli hosts were used to determine the optimal expression system. Expression was highest with the pET22b(+) vector and the Rosetta(DE3)pLysS host strain, which produced approximately 60 mg purified His-tagged scFv per liter of culture (3.3 g wet weight cells). Although the production of soluble scFv was preferred, overproduced scFv formed inclusion bodies under every expression condition. Therefore, inclusion bodies had to be isolated, washed, solubilized, and refolded. The FoldIt protein refolding kit and enzyme-linked immunosorbent assay were sequentially used to determine the optimal refolding conditions that would produce active His-tagged scFv. Immobilized metal affinity chromatography was used for the final purification of the refolded active scFv. The polyol-responsiveness of the scFv was determined by an ELISA-elution assay. Although the scFv loses considerable affinity for its antigen, it maintains similar polyol-responsiveness as the parent monoclonal antibody, PR-mAb NT73.

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.

Identification of Sp2 as a transcriptional repressor of carcinoembryonic antigen-related cell adhesion molecule 1 in tumorigenesis.

Down-regulation of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) tumor suppressor gene expression is common in several malignancies including prostate, colon, and breast cancer. The mechanism that mediates this down-regulation is not known. Here, we report that down-regulation of CEACAM1 expression in prostate cancer cells occurs primarily at the transcriptional level and is mediated by Sp2, a member of the Sp family of transcription factors. Sp2 binds to the CEACAM1 promoter in vitro and in vivo, and transient overexpression of Sp2 down-regulates endogenous CEACAM1 expression in normal prostate epithelial cells. Sp2 appears to repress CEACAM1 gene expression by recruiting histone deacetylase activity to the CEACAM1 promoter. In human prostate cancer specimens, Sp2 expression is high in prostate cancer cells but low in normal prostate epithelial cells and is inversely correlated with CEACAM1 expression. Our studies show that transcriptional repression by Sp2 represents one mechanism by which CEACAM1 tumor suppressor gene is down-regulated in prostate cancer.

An epitope tag derived from human transcription factor IIB that reacts with a polyol-responsive monoclonal antibody.

Polyol-responsive monoclonal antibodies (PR-mAbs) provide a strategy to purify active, nondenatured proteins by a single-step immunoaffinity chromatography procedure. The high affinity interaction between these antibodies and the antigen can be dissociated in the presence of a nonchaotropic salt and a low molecular weight polyhydroxylated compound (polyol). The epitope for PR-mAb IIB8 is located near the N-terminus of the human transcription factor IIB (TFIIB). The epitope is an eight amino acid sequence, TKDPSRVG, that can be fused to a desired protein for use as a purification tag. This epitope tag (termed hIIB) was fused to the C-terminus of green fluorescent protein (GFP). An additional GFP fusion protein utilized another version of hIIB containing a point mutation at position two. These fusion proteins, expressed in Escherichia coli, allowed successful separation of the desired protein in a single chromatographic step. This strategy extends PR-mAb gentle-release purification to numerous expressed proteins.

Development of an epitope tag for the gentle purification of proteins by immunoaffinity chromatography: application to epitope-tagged green fluorescent protein.

Polyol-responsive monoclonal antibodies (mAbs) are useful tools for the gentle purification of proteins and protein complexes. These are high-affinity mAbs that release the antigen in the presence of a nonchaotropic salt and a low-molecular-weight polyhydroxylated compound (polyol). The epitope for the polyol-responsive mAb NT73, which reacts with Escherichia coli RNA polymerase, was located at the C terminus of the beta' subunit. Using recombinant DNA techniques, we have identified the epitope to be within the 13-amino-acid sequence SLAELLNAGLGGS and have developed an epitope tag that can be fused to a protein of interest for use as a purification tag. This epitope tag (designated Softag1) was fused to either the N or the C terminus of the green fluorescent protein. These tagged proteins were expressed in E. coli, and the tagged proteins were purified from the soluble fraction by a single-step immunoaffinity chromatography procedure. This approach extends the powerful technique of gentle-release immunoaffinity chromatography to many expressed proteins.

A cross-reactive polyol-responsive monoclonal antibody useful for isolation of core RNA polymerase from many bacterial species.

The use of antibodies for protein purification is a powerful technique but the release of the target protein in its active form is often difficult. So called "polyol-responsive" monoclonal antibodies (PR-MAbs) have a feature that allows elution of the antigen under very gentle conditions, so that even multi-subunit proteins can be released in their active form. In this work a PR-MAb, 8RB13, was isolated that can purify RNA polymerase (RNAP) from many different bacterial species. High specificity towards RNAP with a broad species cross-reactivity was achieved by immunization with RNAP from Escherichia coli and screening with Bacillus subtilis RNA polymerase. The isolated MAb could detect the beta-subunit of RNA polymerase from 10 out of 12 species tested on a Western blot indicating its potential for purification of core RNAP from these organisms. Representatively, four of these species E. coli, B. subtilis, Pseudomonas aeruginosa, and Streptomyces coelicolor were subjected to immunoaffinity purification yielding RNA polymerases that were active in in vitro transcription and seemed to be primarily core polymerase, lacking sigma-subunits.

Advances in gentle immunoaffinity chromatography.

Immunoaffinity chromatography is one of the most powerful fractionation steps available for protein purification; however, it is often difficult to elute bound protein without using harsh or denaturing elution conditions. The development of methods to identify monoclonal antibodies that bind antigens tightly, but release under gentle, non-denaturing conditions has made possible the immunoaffinity purification of labile, multisubunit enzyme complexes with high yield and high specific activity. This work has implications for emerging proteomic applications, allowing identification of new protein-protein interaction partners, retention of biological activity and the isolation of protein complexes more amenable to crystallization and structure determination.