Evaluation of the Homogenous Mobility Shift Assay for Infliximab and Adalimumab Anti-drug Antibody Detection in the Clinical Laboratory.

BACKGROUND: Detecting antidrug antibodies (ADAs) against infliximab or adalimumab is useful for therapeutic drug monitoring. Various ADA detection methods exist, and antibody titer is an output in some algorithms. Homogenous mobility shift assay (HMSA) measures relative ADA concentration and determines drug-ADA complex size in vitro. However, the relevance of complex size determination in drug monitoring remains unclear. Hence, the association between complex size, ADA concentration, and sample detectable neutralizing activity was evaluated. METHODS: Sera from infliximab-treated and adalimumab-treated patients who tested positive for ADA in the National Screening Service were analyzed using 3 ADA assays. HMSA determined the relative ADA concentrations and complex sizes, competitive ligand-binding assay evaluated the sample neutralizing capacity, and enzyme-linked immunosorbent assay detected immunoglobulin (Ig)G4 ADA. RESULTS: Most ADA-positive samples (>80%) formed drug-ADA dimer complexes, whereas 17% had dimer and multimer complexes, and 3% had multimeric complexes. Multimer presence had 100% positive predictive value for detectable neutralizing activity. ADA concentration and detectable neutralizing activity were moderately correlated (r = 0.65) in adalimumab-treated patients and strongly correlated (r = 0.81) in infliximab-treated patients. In adalimumab-treated patients, multimer presence was a stronger predictor of neutralizing activity than ADA concentration was, but not in infliximab-treated patients. However, in infliximab-treated patient samples, multimer presence revealed a distinct subset with high ADA concentrations, neutralizing activity, and IgG4 ADA. CONCLUSIONS: Multimers detected using HMSA had a strong positive predictive value for competitive ligand-binding assay detectable neutralizing activity. Multimeric IgG4-containing ADA-drug complexes revealed a distinct subset of infliximab-treated patient samples, whose clinical relevance merits further investigation.

Directed evolution of orthogonal RNA-RBP pairs through library-vs-library in vitro selection.

RNA-binding proteins (RBPs) and their RNA ligands play many critical roles in gene regulation and RNA processing in cells. They are also useful for various applications in cell biology and synthetic biology. However, re-engineering novel and orthogonal RNA-RBP pairs from natural components remains challenging while such synthetic RNA-RBP pairs could significantly expand the RNA-RBP toolbox for various applications. Here, we report a novel library-vs-library in vitro selection strategy based on Phage Display coupled with Systematic Evolution of Ligands by EXponential enrichment (PD-SELEX). Starting with pools of 1.1 × 1012 unique RNA sequences and 4.0 × 108 unique phage-displayed L7Ae-scaffold (LS) proteins, we selected RNA-RBP complexes through a two-step affinity purification process. After six rounds of library-vs-library selection, the selected RNAs and LS proteins were analyzed by next-generation sequencing (NGS). Further deconvolution of the enriched RNA and LS protein sequences revealed two synthetic and orthogonal RNA-RBP pairs that exhibit picomolar affinity and >4000-fold selectivity.

Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity.

Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the enzymatic activity, specificity and kinetic parameters. Most notably, the separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique, in terms of both time and resources, to high-performance anion exchange chromatography and NMR-based methods, which are the two current 'gold standards' for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and will support the search for small molecule inhibitors of these disease-associated enzymes.

NAC-type transcription factors regulate accumulation of starch and protein in maize seeds.

Grain starch and protein are synthesized during endosperm development, prompting the question of what regulatory mechanism underlies the synchronization of the accumulation of secondary and primary gene products. We found that two endosperm-specific NAC transcription factors, ZmNAC128 and ZmNAC130, have such a regulatory function. Knockdown of expression of ZmNAC128 and ZmNAC130 with RNA interference (RNAi) caused a shrunken kernel phenotype with significant reduction of starch and protein. We could show that ZmNAC128 and ZmNAC130 regulate the transcription of Bt2 and then reduce its protein level, a rate-limiting step in starch synthesis of maize endosperm. Lack of ZmNAC128 and ZmNAC130 also reduced accumulation of zeins and nonzeins by 18% and 24% compared with nontransgenic siblings, respectively. Although ZmNAC128 and ZmNAC130 affected expression of zein genes in general, they specifically activated transcription of the 16-kDa γ-zein gene. The two transcription factors did not dimerize with each other but exemplified redundancy, whereas individual discovery of their function was not amenable to conventional genetics but illustrated the power of RNAi. Given that both the Bt2 and the 16-kDa γ-zein genes were activated by ZmNAC128 or ZmNAC130, we could identify a core binding site ACGCAA contained within their target promoter regions by combining Dual-Luciferase Reporter and Electrophoretic Mobility Shift assays. Consistent with these properties, transcriptomic profiling uncovered that lack of ZmNAC128 and ZmNAC130 had a pleiotropic effect on the utilization of carbohydrates and amino acids.

A possible role of gas-phase electrophoretic mobility molecular analysis (nES GEMMA) in extracellular vesicle research.

The emerging role of extracellular vesicles (EVs) as biomarkers and their envisioned therapeutic use require advanced techniques for their detailed characterization. In this context, we investigated gas-phase electrophoresis on a nano electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA, aka nES differential mobility analyzer, nES DMA) as an alternative to standard analytical techniques. In gas-phase electrophoresis, single-charged, surface-dry, native, polydisperse, and aerosolized analytes, e.g., proteins or bio-nanoparticles, are separated according to their electrophoretic mobility diameter, i.e., globular size. Subsequently, monodisperse particles are counted after a nucleation step in a supersaturated atmosphere as they pass a focused laser beam. Hence, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU from October 18th, 2011). Smaller sample constituents (e.g., co-purified proteins) can be detected next to larger ones (e.g., vesicles). Focusing on platelet-derived EVs, we compared different vesicle isolation techniques. In all cases, nanoparticle tracking analysis (NTA) confirmed the presence of vesicles. However, nES GEMMA often revealed a significant co-purification of proteins from the sample matrix, precluding gas-phase electrophoresis of less-diluted samples containing higher vesicle concentrations. Therefore, mainly peaks in the protein size range were detected. Mass spectrometry revealed that these main contaminants belonged to the group of globulins and coagulation-related components. An additional size exclusion chromatography (SEC) step enabled the depletion of co-purified, proteinaceous matrix components, while a label-free quantitative proteomics approach revealed no significant differences in the detected EV core proteome. Hence, the future in-depth analysis of EVs via gas-phase electrophoresis appears feasible. Platelet-derived extracellular vesicles (EVs)with/without additional size exclusion chromatographic (SEC) purification were subjected to nanoparticle tracking analysis (NTA) and gas-phase electrophoresis (nES GEMMA). The latter revealed presence of co-purified proteins, targetable via mass spectrometry (MS). MS also revealed that SEC did not influence EV protein content. To conclude, nES GEMMA is a valuable tool for quality control of EV-containing samples under native conditions allowing for detection of co-purified proteins from complex matrices.

Methods for detection and study of virus-derived small RNAs produced from the intramolecular base-pairing region of the picornavirus genome.

There is conclusive evidential support for the existence of virus-derived small RNA (vsRNA) in mammals. Two types of vsRNA have been reported from picornaviruses. The first is virus-derived short-interfering RNA (vsiRNA) that is processed from viral double-stranded RNA intermediates during RNA replication. The other is small RNA derived from the highly base-paired single-stranded genomic region, e.g. the internal ribosome entry site (IRES) of picornaviruses. vsiRNA interacts with the Argonaute protein to control viral RNA replication through the process of RNA interference. However, the function of structure-based vsRNA is largely unknown. We previously identified vsRNA1 generated from the enterovirus-A71 (EV-A71) IRES region by the endogenous enzyme Dicer. Exogenous vsRNA1 can inhibit IRES activity both in vivo and in vitro, hence viral replication is inhibited. Here we describe key methods used to characterize vsRNA, including annotation by next-generation sequencing, abundance measurement by Northern blotting, determination of Dicer-dependence by gel-shift assay and in vitro cleavage assay, and the inhibitory effect on IRES activity via in vitro translation assay.

Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the transcriptional regulator Lsr2 at threonine 112.

Mycobacterium tuberculosis (Mtb) is able to persist in the body through months of multi-drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the protein kinase B (PknB) which controls peptidoglycan biosynthesis during growth. Here, we observed that depletion of PknB resulted in specific transcriptional changes that are likely caused by reduced phosphorylation of the H-NS-like regulator Lsr2 at threonine 112. The activity of PknB towards this phosphosite was confirmed with purified proteins, and this site was required for adaptation of Mtb to hypoxic conditions, and growth on solid media. Like H-NS, Lsr2 binds DNA in sequence-dependent and non-specific modes. PknB phosphorylation of Lsr2 reduced DNA binding, measured by fluorescence anisotropy and electrophoretic mobility shift assays, and our NMR structure of phosphomimetic T112D Lsr2 suggests that this may be due to increased dynamics of the DNA-binding domain. Conversely, the phosphoablative T112A Lsr2 had increased binding to certain DNA sites in ChIP-sequencing, and Mtb containing this variant showed transcriptional changes that correspond with the change in DNA binding. In summary, PknB controls Mtb growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2.

Complex between a Multicrossover DNA Nanostructure, PX-DNA, and T7 Endonuclease I.

Paranemic crossover DNA (PX-DNA) is a four-stranded multicrossover structure that has been implicated in recombination-independent recognition of homology. Although existing evidence has suggested that PX is the DNA motif in homologous pairing (HP), this conclusion remains ambiguous. Further investigation is needed but will require development of new tools. Here, we report characterization of the complex between PX-DNA and T7 endonuclease I (T7endoI), a junction-resolving protein that could serve as the prototype of an anti-PX ligand (a critical prerequisite for the future development of such tools). Specifically, nuclease-inactive T7endoI was produced and its ability to bind to PX-DNA was analyzed using a gel retardation assay. The molar ratio of PX to T7endoI was determined using gel electrophoresis and confirmed by the Hill equation. Hydroxyl radical footprinting of T7endoI on PX-DNA is used to verify the positive interaction between PX and T7endoI and to provide insight into the binding region. Cleavage of PX-DNA by wild-type T7endoI produces DNA fragments, which were used to identify the interacting sites on PX for T7endoI and led to a computational model of their interaction. Altogether, this study has identified a stable complex of PX-DNA and T7endoI and lays the foundation for engineering an anti-PX ligand, which can potentially assist in the study of molecular mechanisms for HP at an advanced level.

Detection of RNA-protein interactions using a highly sensitive non-radioactive electrophoretic mobility shift assay.

Electrophoretic mobility shift assay (EMSA) is a sensitive technique useful in the identification and characterization of protein interactors with nucleic acids. This assay provides an efficient method to study DNA or RNA binding proteins and to identify nucleic acid substrates. The specific interaction plays important roles in many biological processes such as transcription, translation, splicing, and global gene expression. In this article, we have modified the EMSA technique and developed a non-radioactive straightforward method to study and determine RNA-protein interactions. The labeling of target RNAs by 3'-end biotinylation and the detection of biotin reactivity to streptavidin-conjugated horseradish peroxidase is a highly sensitive approach capable to detect the formation of RNA-protein complexes. Overall, we provide a complete technical guide useful to determine in vitro RNA-protein interactions and analyze RNA target specificity.

Single-cell mobility shift electrophoresis reports protein localization to the cell membrane.

While profiling of cell surface receptors grants valuable insight on cell phenotype, surface receptors alone cannot fully describe activated downstream signaling pathways, detect internalized receptor activity, or indicate constitutively active signaling in subcellular compartments. To measure surface-bound and intracellular targets in the same cell, we introduce a tandem single-cell assay that combines immunofluorescence of surface-bound epithelial cellular adhesion molecule (EpCAM) with subsequent protein polyacrylamide gel electrophoresis (PAGE) of unfixed MCF7 breast cancer cells. After surface staining and cell lysis, surface EpCAM is analyzed by single-cell PAGE, concurrent with immunoprobing of intracellular targets. Consequently, the single-cell electrophoresis step reports localization of both surface and intracellular targets. Unbound intracellular EpCAM is readily resolved from surface EpCAM immunocomplex owing to a ∼30% mobility shift. Flow cytometry and immunofluorescence are in concordance with single-cell PAGE. Lastly, we challenged the stability of the EpCAM immunocomplexes by varying ionic and non-ionic component concentrations in the lysis buffer, the lysis time, and electrophoresis duration. As expected, the harsher conditions proved most disruptive to the immunocomplexes. The compatibility of live-cell immunostaining with single-cell PAGE eliminates the need to perform single-cell imaging by condensing read-out of both surface-bound proteins (as low mobility immune complexes) and intracellular targets to a single immunoblot, thus linking cell type and state.

The effects of IAM38 blocking or CD4 blocking on the binding of exogenous DNA in rabbit sperm.

The binding of exogenous DNA to sperm is a key process for sperm-mediated gene transfer; however, the underlying molecular mechanisms have yet to be elucidated. In the present study, we aimed to identify the DNA binding proteins (DBPs) in rabbit sperm and to gain further understanding of the molecular mechanism of sperm and exogenous DNA interaction. Native polyacrylamide gel electrophoresis was used for separating free sperm proteins and complexes of DNA fragment/sperm proteins. A distinct band was found after Coomassie blue staining, and seven potential proteins were identified by mass spectrometry analysis. An analysis of the physical/chemical properties of the seven proteins revealed that the sperm inner acrosomal membrane protein IAM38 (IAM38) matched the features of the DBPs. Western blotting analysis showed that the IAM38 and CD4 were present in the sperm but not in the seminal plasma. Blocking of the IAM38 impaired the DNA-binding capacity of the sperm. Blocking the CD4 decreased the DNA-uptake capacity of the sperm but did not influence the DNA-binding capacity of the sperm. Moreover, the EGFP-positive embryos and EGFP-positive blastocysts were also decreased after IAM38 blocking or CD4 blocking in comparison with the control group. In conclusion, our results imply that foreign DNA first binds to the transmembrane IAM38 of the sperm plasma membrane and then forms the complex of DNA/IAM38/CD4 with CD4 to complete the transportation of exogenous DNA into the nucleus of sperm.

Discrimination of Anti-drug Antibodies With Neutralizing Capacity in Infliximab- and Adalimumab-Treated Patients: Comparison of the Homogeneous Mobility Shift Assay and the Affinity Capture and Elution Assay.

BACKGROUND: The measurement of anti-drug antibody (ADA) levels in adalimumab (ADAL)-treated and infliximab (IFX)-treated patients is critical for guiding therapeutic strategies. The homogeneous mobility shift assay (HMSA) and affinity capture elution (ACE) assay provide effective, drug-tolerant formats for measuring total ADA levels. However, their ability to discriminate between ADA from samples with or without neutralizing capacity is unclear and therefore was analyzed in this study. METHODS: Sera from ADAL and IFX patients with low drug levels (<1 mcg/mL) were analyzed by ACE, HMSA, and bridging assay. Neutralizing capacity was determined by competitive ligand-binding assay. RESULTS: HMSA and ACE detected high ADA levels in all ADAL (19/42) and IFX (27/64) samples with neutralizing capacity. ADA was also detected in most of the samples without neutralizing capacity, but levels were significantly lower (P < 0.0001). Receiver operator characteristic curve analysis demonstrated that for both assays, ADA levels were a strong discriminatory marker of neutralizing ADA (area under the curve > 0.9, P < 0.0001). Using a signal >8× background as a cut-point, neutralizing ADA could be identified with high specificity (HMSA > 95%, ACE > 85%) and sensitivity (HMSA > 70%, ACE > 80%). The detection of multimeric drug-ADA complexes after HMSA was also a highly specific marker (specificity > 95%) of neutralizing ADA in both ADAL and IFX patients. Results using ACE and HMSA were highly correlated. CONCLUSIONS: Results obtained after HMSA and ACE analysis are strongly correlated, and in both assays, high ADA levels are a specific marker of neutralizing capacity. The detection of multimeric complexes by HMSA also selectively identifies sera with neutralizing capacity. These data support the use of these assays as quantitative rather than simple qualitative measures of ADA.

Predicting Electrophoretic Mobility of Tryptic Peptides for High-Throughput CZE-MS Analysis.

A multiparametric sequence-specific model for predicting peptide electrophoretic mobility has been developed using large-scale bottom-up proteomic CE-MS data (5% (∼0.8M) acetic acid as background electrolyte). Peptide charge (Z) and size (molecular mass, M) are the two major factors determining electrophoretic mobility, in complete agreement with previous studies. The extended size of the data set (>4000 peptides) permits access to many sequence-specific factors that impact peptide mobility. The presence of acidic residues Asp and Glu near the peptide N-terminus is by far the most prominent among them. The induction effect of the side chain of N-terminal Asp reduces the basicity of the N-terminal amino group and, as hence, its charge, by ∼0.27 units, lowering mobility. The correlation of the model (R2 ∼ 0.995) indicates that the peptide separation process in CZE is relatively simple and can be predicted to a much higher precision than current RP-HPLC models. Similar to RP-HPLC prediction studies, we anticipate future developments that introduce peptide migration standards, collect larger data sets for modeling through the alignment of multiple CZE-MS acquisitions, and study of the behavior of peptides carrying post-translational modifications. The increased size of data sets will also permit investigation of the fine-scale effects of peptide secondary structure on peptide mobility. We observed that peptides with higher helical propensity tend to have higher than predicted electrophoretic mobility; the incorporation of these features into CZE migration models will require significantly larger data sets.

A mobility shift assay for DNA detection using nanochannel gradient electrophoresis.

Conventional detection of pathogenic or other biological contamination relies on amplification of DNA using sequence-specific primers. Recent work in nanofluidics has shown very high concentration enhancement of biomolecules with some degree of simultaneous separation. This work demonstrates the combination of these two approaches by selectively concentrating a mobility-shifted hybridization product, potentially enabling rapid detection of rare DNA fragments such as highly specific 16S ribosomal DNA. We have performed conductivity gradient electrofocusing within nanofluidic channels and have shown concentration of hybridized peptide nucleic acids and DNA oligomers. We also show selectivity to single base-pair mismatch on 18-mer oligos. This approach may enable sensitive optical detection of small amounts of DNA.

Simultaneous targeted inhibition of Sox2-Oct4 transcription factors using decoy oligodeoxynucleotides to repress stemness properties in mouse embryonic stem cells.

Transcriptional master regulators like Sox2 and Oct4, which are expressed in various human tumors, have been shown to cause tumor growth promotion as well as epithelial dysplasia by means of interfering with progenitor cell differentiation. In order to investigate the potential of Sox2-Oct4 transcription factor decoy (TFD) strategy for differentiation therapy, mouse embryonic stem cells (mESCs) were used in this study as a model of cancer stem cells (CSCs). Sox2-Oct4 complex decoy ODNs (cd-ODNs) were designed according to their elements in the promoter region of Sox2 gene. DNA-protein interactions between decoy ODNs and their corresponding proteins were examined by electrophoretic mobility shift assay (EMSA). Then, decoy and scrambled ODNs were transfected into mESCs with lipofectamine under 2 inhibitors (2i) conditions. Fluorescence and confocal microscopy, cell viability, cell cycle and apoptosis analysis, alkaline phosphatase, embryoid body formation assay, and real-time PCR were used to conduct further investigations. EMSA data showed that Sox2-Oct4 decoy ODNs bound specifically to their recombinant proteins. The results revealed that the synthesized complex decoy can concomitantly target Sox2 and Oct4, which subsequently represses the stemness properties of mESCs compared to controls through decreasing cell viability, arresting cell cycle in G0 /G1 phases, inducing apoptosis, and modulating differentiation in mESCs despite the presence of 2i/LIF in cell culture. While cd-ODN strategy seems to offer great promise for cancer therapy, further studies are still required to put this powerful investigative tool in practice for a wide range of human cancers.

Surface coating and matrix effect on the electrophoretic mobility of gold nanoparticles: a capillary electrophoresis-inductively coupled plasma mass spectrometry study.

Capillary electrophoresis (CE) is considered as a versatile technique in the size-based separation and speciation of nanomaterials. The electrophoretic mobility is determined by charge and size of an analyte which are affected by the surface composition of nanomaterials. Size-dependent differential electrophoretic mobility is used as a mechanism for size-based separation of nanoparticles. Understanding the effect of surface chemistry on the electrophoretic mobility of nanomaterials in CE is critical in obtaining accurate results in retention-based size calculation. A suite of gold nanoparticles (NPs) varied in sizes with different coatings, including citric acid (CA), lipoic acid (LA), tannic acid (TA), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), branched polyethyleneimine (BPEI), and bovine serum albumin (BSA), were selected to evaluate their impact to the migration pattern of gold NPs. Additionally, surface-coated gold NPs dispersed in Suwannee River humic acid (SRHA) solution and fetal bovine serum (FBS) were used to investigate the matrix effect. It was found that the correlation between NP size and relative electrophoretic mobility is highly dependent on the capping agents. The matrix component in the SRHA solution only exhibited limited influence to the migration of NPs while electrophoretic behaviors were drastically altered in the presence of FBS matrix.

A microfluidics-based mobility shift assay to identify new inhibitors of ß-secretase for Alzheimer's disease.

The ß-secretase (BACE1) initiates the generation of toxic amyloid-ß peptide (Aß) from amyloid-ß precursor protein (APP), which was widely considered to play a key role in the pathogenesis of Alzheimer's disease (AD). Here, a novel microfluidics-based mobility shift assay (MMSA) was developed, validated, and applied for the screening of BACE1 inhibitors for AD. First, the BACE1 activity assay was established with a new fluorescent peptide substrate (FAM-EVNLDAEF) derived from the Swedish mutant APP, and high-quality ratiometric data were generated in both endpoint and kinetic modes by electrophoretic separation of peptide substrate from the BACE1 cleaved product (FAM-EVNL) before fluorescence quantification. To validate the assay, the inhibition and kinetic parameter values of two known inhibitors (AZD3839 and AZD3293) were evaluated, and the results were in good agreement with those reported by other methods. Finally, the assay was applied to screen for new inhibitors from a 900-compound library in a 384-well format, and one novel hit (IC50 = 26.5 ± 1.5 µM) was identified. Compared with the common fluorescence-based assays, the primary advantage of the direct MMSA was to discover novel BACE1 inhibitors with lower auto-fluorescence interference, and its superb capability for kinetic study. Graphical abstract Microfluidics-based mobility shift assay for BACE1.

Assessing 2D electrophoretic mobility spectroscopy (2D MOSY) for analytical applications.

Electrophoretic displacement of charged entity phase modulates the spectrum acquired in electrophoretic NMR experiments, and this modulation can be presented via 2D FT as 2D mobility spectroscopy (MOSY) spectra. We compare in various mixed solutions the chemical selectivity provided by 2D MOSY spectra with that provided by 2D diffusion-ordered spectroscopy (DOSY) spectra and demonstrate, under the conditions explored, a superior performance of the former method. 2D MOSY compares also favourably with closely related LC-NMR methods. The shape of 2D MOSY spectra in complex mixtures is strongly modulated by the pH of the sample, a feature that has potential for areas such as in drug discovery and metabolomics. Copyright © 2016 The Authors. Magnetic Resonance in Chemistry published by John Wiley & Sons Ltd.

Transcriptional Repression of the VC2105 Protein by Vibrio FadR Suggests that It Is a New Auxiliary Member of the fad Regulon.

UNLABELLED: Recently, our group along with others reported that the Vibrio FadR regulatory protein is unusual in that, unlike the prototypical fadR product of Escherichia coli, which has only one ligand-binding site, Vibrio FadR has two ligand-binding sites and represents a new mechanism for fatty acid sensing. The promoter region of the vc2105 gene, encoding a putative thioesterase, was mapped, and a putative FadR-binding site (AA CTG GTA AGA GCA CTT) was proposed. Different versions of the FadR regulatory proteins were prepared and purified to homogeneity. Both electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) determined the direct interaction of the vc2105 gene with FadR proteins of various origins. Further, EMSAs illustrated that the addition of long-chain acyl-coenzyme A (CoA) species efficiently dissociates the vc2105 promoter from the FadR regulator. The expression level of the Vibrio cholerae vc2105 gene was elevated 2- to 3-fold in a fadR null mutant strain, validating that FadR is a repressor for the vc2105 gene. The ß-galactosidase activity of a vc2105-lacZ transcriptional fusion was increased over 2-fold upon supplementation of growth medium with oleic acid. Unlike the fadD gene, a member of the Vibrio fad regulon, the VC2105 protein played no role in bacterial growth and virulence-associated gene expression of ctxAB (cholera toxin A/B) and tcpA (toxin coregulated pilus A). Given that the transcriptional regulation of vc2105 fits the criteria for fatty acid degradation (fad) genes, we suggested that it is a new member of the Vibrio fad regulon. IMPORTANCE: The Vibrio FadR regulator is unusual in that it has two ligand-binding sites. Different versions of the FadR regulatory proteins were prepared and characterized in vitro and in vivo. An auxiliary fad gene (vc2105) from Vibrio was proposed that encodes a putative thioesterase and has a predicted FadR-binding site (AAC TGG TA A GAG CAC TT). The function of this putative binding site was proved using both EMSA and SPR. Further in vitro and in vivo experiments revealed that the Vibrio FadR is a repressor for the vc2105 gene. Unlike fadD, a member of the Vibrio fad regulon, VC2105 played no role in bacterial growth and expression of the two virulence-associated genes (ctxAB and tcpA). Therefore, since transcriptional regulation of vc2105 fits the criteria for fad genes, it seems likely that vc2105 acts as a new auxiliary member of the Vibrio fad regulon.

Photoaffinity electrophoretic mobility shift assay using photoreactive DNA bearing 3-trifluoromethyl-3-phenyldiazirine in its phosphate backbone.

Photoaffinity cross-linking enables the analysis of interactions between DNA and proteins even under denaturing conditions. We present a photoaffinity electrophoretic mobility shift assay (EMSA) in which two heterogeneous techniques-photoaffinity cross-linking using DNA bearing 3-trifluoromethyl-3-phenyldiazirine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis-are combined. To prepare the photoreactive DNA, which is an essential tool for photoaffinity EMSA, we first determined the optimal conditions for the integration of 4-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl bromide to the specific site of oligonucleotide where phosphodiester linkage was replaced with phosphorothioate linkage. The photoaffinity EMSA was developed using the POU (initial letters of three genes: Pit-l, Oct-1,2, and unc-86) domain region of Oct-1 protein, which specifically bound to octamer DNA motif (ATGCAAAT). The affinity-purified recombinant POU domain proteins conjugated with glutathione-S-transferase (GST) contained three distinct proteins with molecular weights of 34, 36, and 45 kDa. The photoaffinity EMSA could clearly distinguish the individual binding abilities of three proteins on a single lane and showed that the whole POU domain protein specifically bound to octamer DNA motif by competition experiments. Using the nuclear extract of HeLa cells, the photoaffinity EMSA revealed that at least five specific proteins could bind to the octamer DNA motif. These results show that photoaffinity EMSA using 3-trifluoromethyl-3-phenyldiazirine can provide high-performance analysis of DNA-binding proteins.