A novel expression system for production of soluble prion proteins in E. coli

Expression of eukaryotic proteins in Escherichia coli is challenging, especially when they contain disulfide bonds. Since the discovery of the prion protein (PrP) and its role in transmissible spongiform encephalopathies, the need to obtain large quantities of the recombinant protein for research purposes has been essential. Currently, production of recombinant PrP is achieved by refolding protocols. Here, we show that the co-expression of two different PrP with the human Quiescin Sulfhydryl OXidase (QSOX), a human chaperone with thiol/disulfide oxidase activity, in the cytoplasm of E. coli produces soluble recombinant PrP. The structural integrity of the soluble PrP has been confirmed by nuclear magnetic resonance spectroscopy, demonstrating that properly folded PrP can be easily expressed in bacteria. Furthermore, the soluble recombinant PrP produced with this method can be used for functional and structural studies

Etude moléculaire de la régulation du catabolisme anaérobique de l'arginine chez Bacillus licheniformis

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Purification of ArcR, an oxidation-sensitive regulatory protein from Bacillus licheniformis

In Bacillus licheniformis, ArcR, a transcriptional activator of the Crp/Fnr family, is required for expression of the anaerobic pathway of arginine catabolism, the arginine deiminase pathway. The method described here allows the purification of milligram quantities of functional ArcR from a recombinant Escherichia coli strain. The solubility properties of ArcR were much exploited during the purification process. The protein appeared highly sensitive to oxidation. Oxidation-induced precipitation of the protein was attributed to the formation of intermolecular disulfide bridges. Alkylation of mutant proteins with single substitutions showed that both cysteine residues of the protein, C178 and C205, are involved in formation of the disulfide bridges. Substitution of both cysteines yielded a functional protein insensitive to oxidation and able to form a complex with its cognate target on the DNA. © 2004 Elsevier Inc. All rights reserved.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Structural relationships in the lysozyme superfamily: significant evidence for glycoside hydrolase signature motifs.

Chitin is a polysaccharide that forms the hard, outer shell of arthropods and the cell walls of fungi and some algae. Peptidoglycan is a polymer of sugars and amino acids constituting the cell walls of most bacteria. Enzymes that are able to hydrolyze these cell membrane polymers generally play important roles for protecting plants and animals against infection with insects and pathogens. A particular group of such glycoside hydrolase enzymes share some common features in their three-dimensional structure and in their molecular mechanism, forming the lysozyme superfamily.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Expression and purification in high yield of a functionally active recombinant human Type I inositol(1,4,5)P3 5-phosphatase.

Inositol polyphosphates are the most widespread second messenger molecules in eukaryotic cells. Human Type I inositol 1,4,5-triphosphate (Ins(1,4,5)P(3)) 5-phosphatase removes the D-5 position phosphate from soluble Ins(1,4,5)P(3,) a key event in cell signaling particularly in Ca(2+) homeostasis. In this study, the cDNA encoding human Type I Ins(1,4,5)P(3) 5-phosphatase was subcloned into a modified pMAL expression vector. This plasmid produces a recombinant protein in fusion with affinity tags located at its N-terminus, consisting in a maltose binding protein (MPB) and an octa-histidine stretch. The construction was transformed into Escherichia coli BL21 (DE3) expression strain. This dual tag strategy allows the purification of milligrams of highly purified protein. The recombinant human Type I Ins(1,4,5)P(3) 5-phosphatase is active and can thus be used for functional and structural studies.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Characterization of the PAS domain in the sensor-kinase BvgS: mechanical role in signal transmission.

International audienceBACKGROUND: In bacteria, signal-transduction two-component systems are major players for adaptation to environmental stimuli. The perception of a chemical or physical signal by a sensor-kinase triggers its autophosphorylation. The phosphoryl group is then transferred to the cognate response regulator, which mediates the appropriate adaptive response. Virulence of the whooping cough agent Bordetella pertussis is controlled by the two-component system BvgAS. Atypically, the sensor-kinase BvgS is active without specific stimuli at 37[degree sign]C in laboratory conditions and is inactivated by the addition of negative chemical modulators. The structure of BvgS is complex, with two tandem periplasmic Venus flytrap domains and a cytoplasmic PAS domain that precedes the kinase domain, which is followed by additional phosphotransfer domains. PAS domains are small, ubiquitous sensing or regulatory domains. The function of the PAS domain in BvgS remains unknown. RESULTS: We showed that recombinant BvgS PAS proteins form dimers that are stabilized by alpha helical regions flanking the PAS core. A structural model of the PAS domain dimer was built and probed by site-directed mutagenesis and by biochemical and functional analyses. Although we found no ligands for the PAS domain cavity, its integrity is required for signaling. We also showed that the structural stability of the PAS core and its proper coupling to its flanking N- and C-terminal alpha helices are crucial for BvgS activity. CONCLUSIONS: We propose that a major function of the BvgS PAS domain is to maintain conformational signals arising from mechanical strain generated by the periplasmic domain. The tight structure of the PAS core and its connections with the upstream and downstream helices ensure signaling to the kinase domain, which determines BvgS activity. Many mild substitutions that map to the PAS domain keep BvgS active but make it unresponsive to negative modulators, supporting that modulation increases conformational strain in the protein

Structural analysis of the interaction between spiroisoxazoline SMARt-420 and the Mycobacterium tuberculosis repressor EthR2.

Inhibition of transcriptional regulators of bacterial pathogens with the aim of reprogramming their metabolism to modify their antibiotic susceptibility constitutes a promising therapeutic strategy. One example is the bio-activation of the anti-tubercular pro-drug ethionamide, which activity could be enhanced by inhibiting the transcriptional repressor EthR. Recently, we discovered that inhibition of a second transcriptional repressor, EthR2, leads to the awakening of a new ethionamide bio-activation pathway. The x-ray structure of EthR2 was solved at 2.3 Å resolution in complex with a compound called SMARt-420 (Small Molecule Aborting Resistance). Detailed comparison and structural analysis revealed interesting insights for the upcoming structure-based design of EthR2 inhibitors as an alternative to revert ethionamide resistance in Mycobacterium tuberculosis.SCOPUS: ar.jSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Expression, Purification, Characterization, and Solution Nuclear Magnetic Resonance Study of Highly Deuterated Yeast Cytochrome <i>c</i> Peroxidase with Enhanced Solubility

Here we present the preparation, biophysical characterization, and nuclear magnetic resonance (NMR) spectroscopy study of yeast cytochrome <i>c</i> peroxidase (CcP) constructs with enhanced solubility. Using a high-yield <i>Escherichia coli</i> expression system, we routinely produced uniformly labeled [²H,¹³C,¹⁵N]­CcP samples with high levels of deuterium incorporation (96–99%) and good yields (30–60 mg of pure protein from 1 L of bacterial culture). In addition to simplifying the purification procedure, introduction of a His tag at either protein terminus dramatically increases its solubility, allowing preparation of concentrated, stable CcP samples required for multidimensional NMR spectroscopy. Using a range of biophysical techniques and X-ray crystallography, we demonstrate that the engineered His tags neither perturb the structure of the enzyme nor alter the heme environment or its reactivity toward known ligands. The His-tagged CcP constructs remain catalytically active yet exhibit differences in the interaction with cytochrome <i>c</i>, the physiological binding partner, most likely because of steric occlusion of the high-affinity binding site by the C-terminal His tag. We show that protein perdeuteration greatly increases the quality of the double- and triple-resonance NMR spectra, allowing nearly complete backbone resonance assignments and subsequent study of the CcP by heteronuclear NMR spectroscopy

Ester prodrugs of ciprofloxacin as DNA-gyrase inhibitors: synthesis, antiparasitic evaluation and docking studies

Novel ester prodrugs of ciprofloxacin were synthesized, and tested for their antimalarial and antitoxoplasma activity. These new compounds proved to be extremely efficient against these parasites. Molecular modeling and computational calculations were used to understand the mechanisms of action of these drugs. © The Royal Society of Chemistry 2011.F.I. 3.2320SCOPUS: ar.jinfo:eu-repo/semantics/publishe