Key features of σS required for specific recognition by Crl, a transcription factor promoting assembly of RNA polymerase holoenzyme.

Bacteria use multiple sigma factors to coordinate gene expression in response to environmental perturbations. In Escherichia coli and other γ-proteobacteria, the transcription factor Crl stimulates σ(S)-dependent transcription during times of cellular stress by promoting the association of σ(S) with core RNA polymerase. The molecular basis for specific recognition of σ(S) by Crl, rather than the homologous and more abundant primary sigma factor σ(70), is unknown. Here we use bacterial two-hybrid analysis in vivo and p-benzoyl-phenylalanine cross-linking in vitro to define the features in σ(S) responsible for specific recognition by Crl. We identify residues in σ(S) conserved domain 2 (σ(S)2) that are necessary and sufficient to allow recognition of σ(70) conserved domain 2 by Crl, one near the promoter-melting region and the other at the position where a large nonconserved region interrupts the sequence of σ(70). We then use luminescence resonance energy transfer to demonstrate directly that Crl promotes holoenzyme assembly using these specificity determinants on σ(S). Our results explain how Crl distinguishes between sigma factors that are largely homologous and activates discrete sets of promoters even though it does not bind to promoter DNA.

Artifact-inducing enrichment of ethylenediaminetetraacetic acid and ethyleneglycoltetraacetic acid on anion exchange resins.

Multivalent metal chelators, ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA), are used extensively during protein purification. Both strong (Q) and weak (DEAE) anion exchange resins were found to adsorb surprisingly large quantities of EDTA and EGTA that elute from the resin at NaCl concentrations of approximately 240 mM (EDTA) and 140 mM (EGTA). The EDTA/EGTA elution and saturation parameters were determined for five commonly used anion exchange resins. The resulting concentration of eluted EDTA was 10- to 200-fold higher than that originally present in the sample or in the mobile phase. Samples from fractions containing such a high concentration of EDTA were found to inhibit Mg(2+)-dependent polymerase chain reaction (PCR). EDTA binding to the anion exchange resins could saturate the resin, decrease its binding capacity, and displace weakly bound proteins such as green fluorescent protein (GFP). Several steps are suggested to minimize on-column EDTA concentration, including column equilibration in the absence of any EDTA, lower concentrations (0.1-0.5mM) of EDTA, monitoring eluate absorbance at 280 nm as well as at 215 nm, adding EDTA back into fractions eluting before the EDTA peak, and performing blank column runs to control for the effect of changes in EDTA concentration in downstream assays.

Expression and purification of full-length mouse CARM1 from transiently transfected HEK293T cells using HaloTag technology.

Coactivator-associated arginine methyl transferase 1 (CARM1) is a protein arginine methyltransferase (PRMT) family member that functions as a coactivator in androgen and estrogen signaling pathways and plays a role in the progression of prostate and breast cancer. CARM1 catalyzes methylation of diverse protein substrates. Prior attempts to purify the full-length mouse CARM1 protein have proven unsatisfactory. The full-length protein expressed in Escherichia coli forms insoluble inclusion bodies that are difficult to denature and refold. The presented results demonstrate the use of a novel HaloTag™ technology to purify full-length CARM1 from both E. coli and mammalian HEK293T cells. A small amount of CARM1 was purified from E. coli; however, the protein was truncated on the N-terminus by 10-50 amino acids, most likely due to endogenous proteolytic activity. In contrast, substantial quantities of soluble full-length CARM1 were purified from transiently transfected HEK293T cells. The CARM1 from HEK293T cells was isolated alongside a number of co-purifying interacting proteins. The covalent bond formed between the HaloTag and the HaloLink resin allowed the use of stringent wash conditions without risk of eluting the CARM1 protein. The results also illustrate a highly effective approach for purifying and enriching both CARM1-associated proteins as well as substrates for CARM1's methyltransferase activity.

Expression, purification, and refolding of active Nrf2 transcription factor fused to protein transduction TAT tag.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor responsible for activation of diverse genes that protect the cell against xenobiotics and oxidative stress. The Nrf2-dependent transcription is tightly controlled by cytoplasmic interaction of Nrf2 with its inhibitor, Kelch ECH-associating protein 1 (Keap1). The Keap1-mediated inhibition can be overcome by addition of xenobiotics or by overexpression of Nrf2 protein. The overexpressed Nrf2 overwhelms the Keap1 inhibition, translocates into the nucleus and activates antioxidant response element (ARE)-dependent gene transcription that protects the cell against oxidative stress. We expressed and purified recombinant mouse Nrf2 protein fused to a protein transduction domain (TAT), derived from transactivator of transcription protein from HIV-1. Full-length TAT-Nrf2 was expressed in Escherichia coli in insoluble inclusion bodies and purified to homogeneity using denaturing size exclusion chromatography. Optimal refolding conditions were determined through the use of a light scattering-based refolding assay and analytical size exclusion chromatography. The results demonstrate that the refolded TAT-Nrf2 could transduce into cultured human neuroblastoma cells. The transduced TAT-Nrf2 activated transcription of ARE-dependent genes and conferred protection against intracellular reactive oxygen species (ROS) generated by hydrogen peroxide exposure.

Differential modification of Cys10 alters transthyretin's effect on beta-amyloid aggregation and toxicity.

Tg2576 mice produce high levels of beta-amyloid (Abeta) and develop amyloid deposits, but lack neurofibrillary tangles and do not suffer the extensive neuronal cell loss characteristic of Alzheimer's disease. Protection from Abeta toxicity has been attributed to up-regulation of transthyretin (TTR), a normal component of plasma and cerebrospinal fluid. We compared the effect of TTR purified from human plasma (pTTR) with that produced recombinantly (rTTR) on Abeta aggregation and toxicity. pTTR slowed Abeta aggregation but failed to protect primary cortical neurons from Abeta toxicity. In contrast, rTTR accelerated aggregation, while effectively protecting neurons. This inverse correlation between Abeta aggregation kinetics and toxicity is consistent with the hypothesis that soluble intermediates rather than insoluble fibrils are the most toxic Abeta species. We carried out a detailed comparison of pTTR with rTTR to ascertain the probable cause of these different effects. No differences in secondary, tertiary or quaternary structure were detected. However, pTTR differed from rTTR in the extent and nature of modification at Cys10. We hypothesize that differential modification at Cys10 regulates TTR's effect on Abeta aggregation and toxicity.

Rational development of beta-peptide inhibitors of human cytomegalovirus entry.

Human cytomegalovirus (HCMV) is a pervasive and significant pathogen. At present, there is no HCMV vaccine, and the available drugs target only replication events. Thus, new therapeutic strategies are needed. HCMV fusion appears to require interactions of alpha-helical regions in viral surface glycoproteins gB and gH. Oligomers of beta-amino acids ("beta-peptides") are attractive unnatural scaffolds for mimicry of specific protein surfaces, because beta-peptides adopt predictable helical conformations and resist proteolysis. Here, we report the development of beta-peptides designed to mimic the gB heptad repeat and block HCMV entry. The most potent beta-peptide inhibits HCMV infection in a cell based-assay with an IC50 of approximately 30 microm. Consistent with our structure-based design strategy, inhibition is highly specific for HCMV relative to other related viruses. Mechanistic studies indicate that inhibitory beta-peptides act by disrupting membrane fusion. Our findings raise the possibility that beta-peptides may provide a general platform for development of a new class of antiviral agents and that inhibitory beta-peptides will constitute new tools for elucidating viral entry mechanisms.