Nancy E. Thompson

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.

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.

Immunoaffinity purification of RNA polymerase II and transcription factors using polyol-responsive monoclonal antibodies.

Publisher Summary This chapter discusses the immunoaffinity purification of RNA polymerase II and transcription factors using polyol-responsive monoclonal antibodies. Using these methods, immunoaffinity chromatography procedures are developed for many of the proteins. At least one other research group has been able to use these methods to isolate a polyol-responsive MAb and to develop an immunoaffinity purification procedure for the enzyme. Polyol-responsive MAbs prove to be important tools for the rapid purification of labile proteins by immunoaffinity chromatography. The chapter developed a method of eluting labile proteins from certain MAbs. This elution procedure uses a combination of a low–molecular-weight polyhydroxylated compound (polyol) and a nonchaotropic salt. However, only some MAbs respond to these elution conditions. A method to screen MAbs for the ability to respond to the polyol/salt elution method is developed. This screening method is a modified enzyme-linked immunosorbent assay (ELISA). The ELISA-elution assay can also be used to help optimize the eluting conditions.

The HIP1 initiator element plays a role in determining the in vitro requirement of the dihydrofolate reductase gene promoter for the C-terminal domain of RNA polymerase II.

We examined the ability of purified RNA polymerase (RNAP) II lacking the carboxy-terminal heptapeptide repeat domain (CTD), called RNAP IIB, to transcribe a variety of promoters in HeLa extracts in which endogenous RNAP II activity was inhibited with anti-CTD monoclonal antibodies. Not all promoters were efficiently transcribed by RNAP IIB, and transcription did not correlate with the in vitro strength of the promoter or with the presence of a consensus TATA box. This was best illustrated by the GC-rich, non-TATA box promoters of the bidirectional dihydrofolate reductase (DHFR)-REP-encoding locus. Whereas the REP promoter was transcribed by RNAP IIB, the DHFR promoter remained inactive after addition of RNAP IIB to the antibody-inhibited reactions. However, both promoters were efficiently transcribed when purified RNAP with an intact CTD was added. We analyzed a series of promoter deletions to identify which cis elements determine the requirement for the CTD of RNAP II. All of the promoter deletions of both DHFR and REP retained the characteristics of their respective full-length promoters, suggesting that the information necessary to specify the requirement for the CTD is contained within approximately 65 bp near the initiation site. Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro. Since the synthetic minimal promoter of DHFR and the smallest REP promoter deletion are both activated by Sp1, the differential response in this assay does not result from upstream activators. However, the sequences around the start sites of DHFR and REP are not similar and our data suggest that they bind different proteins. Therefore, we propose that specific initiator elements are important for determination of the requirement of some promoters for the CTD.

Expression, Purification of, and Monoclonal Antibodies to σ Factors from Escherichia coli

Publisher Summary This chapter explores the escherichia coli DNA-dependent RNA polymerase that is the sole enzyme responsible for the synthesis of messenger, transfer, and ribosomal RNA. The E. Coli housekeeping σ factor, σ 70 , was the first prokaryotic σ factor to be purified and characterized. Since then, six additional σ factors have been found in E. Coli K12. All seven sigma factors have been categorized in two families by means of sequence similarity. In addition to unique spacing requirements within promoters, each sigma factor recognizes specific promoter sequences, allowing E. coli the means to regulate gene expression. The major sigma factors of E. coli can be purified using the basic principles of overexpression, isolation of inclusion bodies, denaturation and protein refolding, and purification over an ion-exchange column. Their significant negative charge at physiological pH allows the purification on anion-exchange resin such as PorosHQ or Mono Q resins. The chapter reviews protocols describing the purification of these sigma factors that have been published previously. Therefore, the purification protocols described in this chapter merely summarize, and refine the established protocols using the most current chromatography techniques.

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.

Monoclonal Antibodies to the Enterotoxins and to the Toxic Shock Syndrome Toxin Produced by Staphylococcus aureus

Publisher Summary This chapter presents monoclonal antibodies (MAbs) to the enterotoxins and to the toxic shock syndrome toxin produced by Staphylococcus aureus. Staphylococcus aureus is an important opportunistic pathogen common to humans and many other warm-blooded animals. It is the etiological agent in a variety of pathological conditions ranging from common skin disorders to the life-threatening illness known as toxic shock syndrome. Many healthy individuals carry strains of Staphylococcus aureus as part of the normal microbial population of their nares, pharynx, vagina, and skin of the axilla with no detrimental effects. Some strains of Staphylococcus aureus produce one or more proteins that are classified as toxins by virtue of their destructive biological activities on animals or animal cells. These toxins include the exfoliative toxins, the hemolysins, leukocidin, and the enterotoxins. This chapter describes enterotoxins, the causative agent of staphylococcal food poisoning, and the enterotoxin-like protein associated with strains isolated from patients afflicted with toxic shock syndrome. It also discusses the procedure for isolating MAbs reactive with enterotoxin-like protein and adapting the antibodies to systems for assay and purification of the toxins.

The ABL-MYC retrovirus generates antigen-specific plasmacytomas by in vitro infection of activated B lymphocytes from spleen and other murine lymphoid organs

ABL-MYC is a recombinant retrovirus that constitutively expresses the v-abl and c-myc oncogenes. When used to infect immunized mice this virus rapidly and efficiently induces plasmacytomas of which an unusually high percentage secrete antigen (Ag)-specific monoclonal antibodies. These findings suggested that ABL-MYC targets Ag-stimulated B cells for transformation and that infection of lymphoid cells in vitro might be a useful, alternative method for generating monoclonal, Ag-specific plasmacytomas (ASPCTs). Therefore, we used helper virus-free ABL-MYC to infect suspensions of cells from spleens and other lymphoid organs from mice that had been immunized with a variety of Ags and transplanted them into naive mice. The results show that ABL-MYC preferentially transforms splenocytes that are Ag-reactive. They also demonstrate that ASPCTs can be produced by in vitro infection of cell suspensions from the spleen, lymph nodes and Peyers patches of mice that had been immunized intraperitoneally with sheep red blood cells, Escherichia coli core RNA polymerase or Epstein-Barr virus gp340 protein or immunized orally with live Giardia lamblia parasites. The ASPCTs usually consisted of one to three colnes, secreted antibodies that were quantitatively and qualitatively similar to those obtained from hybridomas, and could continue to secrete Ag-reactive antibody over eight transplant generations.

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.

Applications of Monoclonal Antibodies to the Study of Eukaryotic RNA Polymerases

Transcription is a complex series of carefully regulated events that leads to an RNA product. Central to our understanding of these events is an understanding of how the RNA polymerase (RNAP) interacts with DNA sequences, protein factors, nucleoside triphosphates, and, perhaps, the nuclear suprastructure to faithfully accomplish transcription. Despite the central role that RNAPs play in gene expression, surprisingly little information is available on the mechanisms of these enzymes.