A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin–CDK complex

A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This ‘CHH’ MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2–Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.

Systematic analysis of protein complexes involved in the human RNA polymerase II machinery

La transcription, la maturation d’ARN, et le remodelage de la chromatine sont tous des processus centraux dans l'interprétation de l'information contenue dans l’ADN. Bien que beaucoup de complexes de protéines formant la machinerie cellulaire de transcription aient été étudiés, plusieurs restent encore à identifier et caractériser. En utilisant une approche protéomique, notre laboratoire a purifié plusieurs composantes de la machinerie de transcription de l’ARNPII humaine par double chromatographie d’affinité "TAP". Cette procédure permet l'isolement de complexes protéiques comme ils existent vraisemblablement in vivo dans les cellules mammifères, et l'identification de partenaires d'interactions par spectrométrie de masse. Les interactions protéiques qui sont validées bioinformatiquement, sont choisies et utilisées pour cartographier un réseau connectant plusieurs composantes de la machinerie transcriptionnelle. En appliquant cette procédure, notre laboratoire a identifié, pour la première fois, un groupe de protéines, qui interagit physiquement et fonctionnellement avec l’ARNPII humaine. Les propriétés de ces protéines suggèrent un rôle dans l'assemblage de complexes à plusieurs sous-unités, comme les protéines d'échafaudage et chaperonnes. L'objectif de mon projet était de continuer la caractérisation du réseau de complexes protéiques impliquant les facteurs de transcription. Huit nouveaux partenaires de l’ARNPII (PIH1D1, GPN3, WDR92, PFDN2, KIAA0406, PDRG1, CCT4 et CCT5) ont été purifiés par la méthode TAP, et la spectrométrie de masse a permis d’identifier de nouvelles interactions. Au cours des années, l’analyse par notre laboratoire des mécanismes de la transcription a contribué à apporter de nouvelles connaissances et à mieux comprendre son fonctionnement. Cette connaissance est essentielle au développement de médicaments qui cibleront les mécanismes de la transcription.

Manipulation of the ubiquitin-proteasome system by HIV-1 : role of the accessory protein Vpr

Le virus de l’immunodéficience humaine de type 1 (VIH-1), l’agent étiologique du SIDA, est un rétrovirus complexe arborant plusieurs protéines accessoires : Nef, Vif, Vpr, et Vpu. Celles-ci sont impliquées dans la modulation de la réplication virale, dans l’évasion immunitaire et dans la progression de la pathogenèse du SIDA. Dans ce contexte, il a été démontré que la protéine virale R (Vpr) induit un arrêt de cycle cellulaire en phase G2. Le mécanisme par lequel Vpr exerce cette fonction est l’activation, ATR (Ataxia telangiectasia and Rad3 related)-dépendante, du point de contrôle de dommage à l’ADN, mais les facteurs et mécanismes moléculaires directement impliqués dans cette activité demeurent inconnus. Afin d’identifier de nouveaux facteurs cellulaires interagissant avec Vpr, nous avons utilisé une purification d’affinité en tandem (TAP) pour isoler des complexes protéiques natifs contenant Vpr. Nous avons découvert que Vpr s’associait avec CRL4A(VprBP), un complexe cellulaire d’E3 ubiquitine ligase, comprenant les protéines Cullin 4A, DDB1 (DNA damage-binding protein 1) et VprBP (Vpr-binding protein). Nos études ont mis en évidence que le recrutement de la E3 ligase par Vpr était nécessaire mais non suffisant pour l’induction de l’arrêt de cycle cellulaire en G2, suggérant ainsi que des événements additionnels seraient impliqués dans ce processus. À cet égard, nous apportons des preuves directes que Vpr détourne les fonctions de CRL4A(VprBP) pour induire la polyubiquitination de type K48 et la dégradation protéosomale de protéines cellulaires encore inconnues. Ces événements d’ubiquitination induits par Vpr ont été démontrés comme étant nécessaire à l’activation d’ATR. Finalement, nous montrons que Vpr forme des foyers ancrés à la chromatine co-localisant avec VprBP ainsi qu’avec des facteurs impliqués dans la réparation de l’ADN. La formation de ces foyers représente un événement essentiel et précoce dans l’induction de l’arrêt de cycle cellulaire en G2. Enfin, nous démontrons que Vpr est capable de recruter CRL4A(VprBP) au niveau de la chromatine et nous apportons des preuves indiquant que le substrat inconnu ciblé par Vpr est une protéine associée à la chromatine. Globalement, nos résultats révèlent certains des ménanismes par lesquels Vpr induit des perturbations du cycle cellulaire. En outre, cette étude contribue à notre compréhension de la modulation du système ubiquitine-protéasome par le VIH-1 et son implication fonctionnelle dans la manipulation de l’environnement cellulaire de l’hôte.

Cartographie du réseau d'interactions protéiques de la machinerie de transcription dans les cellules humaines

Les protéines sont les macromolécules les plus polyvalentes de la cellule. Elles jouent un rôle fondamental dans la majorité des processus biologiques à travers la formation de complexes multi-protéiques. Durant la transcription, une multitude de facteurs sont impliquées dans le contrôle de l’activité des complexes ARN polymérases. Notre laboratoire s’est intéressé au réseau d’interaction de la machinerie de transcription des ARN polymérases nucléaires, dans le but de mieux comprendre leurs mécanismes de régulation. Pour ce faire, une procédure protéomique comprenant la purification de complexes protéiques par affinité couplée à la spectrométrie de masse et à l’analyse bioinformatique a été développée. La méthode de purification TAP (Tandem Affinity Purification) a été adaptée pour permettre la purification de complexes protéiques solubles assemblés in vivo à partir de cellules humaines. L’objectif de mon projet de maîtrise était de purifier le complexe de l’ARN Pol I ainsi que de poursuivre l’expansion du réseau d’interactions protéine-protéine de la machinerie de transcription de l’ARN Pol II humaine. À l’aide des protéines POLR1E, TWISTNB, POLR2E, PFDN4, MBD2, XPA, CAND1 et PDCD5 étiquetées (TAP-tag) exprimées dans des lignées cellulaires ECR-293, plusieurs complexes protéiques solubles ont été purifiés et analysés par spectrométrie de masse. Les interactions protéiques ont été triées et validées bioinformatiquement pour donner en final une liste d’interactions ayant un haut degré de confiance à partir de laquelle des réseaux d’interactions protéine-protéine ont été créés. Le réseau créé au cours de ce projet connecte plusieurs composantes de la machinerie transcriptionnelle tels que les ARN Pol I, II et III, les complexes RPAP3/R2TP/prefoldin-like, TRiC/CCT, Mi-2/NuRD et des facteurs de transcription et de réparation de l’ADN. Ce type d’analyse nous a permis d’identifier et de caractériser de nouveaux régulateurs de la machinerie de transcription de l’ARN Pol I et II et de mieux comprendre son fonctionnement.

Étude structure / fonction des sous-unités catalytiques de l'ARN polymérase II

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Fukushima-derived radionuclides in sediments of the Japanese Pacific Ocean coast and various Japanese water samples (seawater, tap water, and coolant water of Fukushima Daiichi reactor unit 5)

We investigated Ocean sediments and seawater from inside the Fukushima exclusion zone and found radiocesium (134Cs and 137Cs) up to 800 Bq kg-1 as well as 90Sr up to 5.6 Bq kg-1. This is one of the first reports on radiostrontium in sea sediments from the Fukushima exclusion zone. Seawater exhibited contamination levels up to 5.3 Bq kg-1 radiocesium. Tap water from Tokyo from weeks after the accident exhibited detectable but harmless activities of radiocesium (well below the regulatory limit). Analysis of the Unit 5 reactor coolant (finding only 3H and even low 129I) leads to the conclusion that the purification techniques for reactor coolant employed at Fukushima Daiichi are very effective.

Bp-13 Pla2: Purification And Neuromuscular Activity Of A New Asp49 Toxin Isolated From Bothrops Pauloensis Snake Venom.

A new PLA2 (Bp-13) was purified from Bothrops pauloensis snake venom after a single chromatographic step of RP-HPLC on μ-Bondapak C-18. Amino acid analysis showed a high content of hydrophobic and basic amino acids and 14 half-cysteine residues. The N-terminal sequence showed a high degree of homology with basic Asp49 PLA2 myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36°-45°C. Full Bp-13 PLA2 activity required Ca(2+); its PLA2 activity was inhibited by Mg(2+), Mn(2+), Sr(2+), and Cd(2+) in the presence and absence of 1 mM Ca(2+). In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent (P < 0.05). Both the replacement of Ca(2+) by Sr(2+) and temperature lowering (24°C) inhibited the Bp-13 PLA2-induced twitch-tension blockade. Bp-13 PLA2 inhibited the contractile response to direct electrical stimulation in curarized mouse PND preparation corroborating its contracture effect. In biventer cervicis preparations, Bp-13 induced irreversible twitch-tension blockade and the KCl evoked contracture was partially, but significantly, inhibited (P > 0.05). The main effect of this new Asp49 PLA2 of Bothrops pauloensis venom is on muscle fiber sarcolemma, with avian preparation being less responsive than rodent preparation. The study enhances biochemical and pharmacological characterization of B. pauloensis venom.

Extraction, purification and biochemical characterization of a peroxidase from Copaifera langsdorffii leaves

The aim of this work is to obtain, purify and characterize biochemically a peroxidase from Copaifera langsdorffii leaves (COP). COP was obtained by acetone precipitation followed by ion-exchange chromatography. Purification yielded 3.5% of peroxidase with the purification factor of 46.86. The COP optimum pH is 6.0 and the temperature is 35 ºC. COP was stable in the pH range of 4.5 to 9.3 and at temperatures below 50.0 ºC. The apparent Michaelis-Menten constants (Km) for guaiacol and H2O2 were 0.04 mM and 0.39 mM respectively. Enzyme turnover was 0.075 s-1 for guaiacol and 0.28 s-1 for hydrogen peroxide. Copaifera langsdorffii leaves showed to be a rich source of active peroxidase (COP) during the whole year. COP could replace HRP, the most used peroxidase, in analytical determinations and treatment of industrial effluents at low cost.

Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (μ = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (μ = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.

A convenient method for lecithin purification from fresh eggs

The increasing demand for fatty acid-free lecithin required modifications in existing purification methods. In this technical note we describe a purification procedure with the following steps: a) homogenization and extraction of yolks obtained from fresh eggs with acetone, b) solubilization with ethanol and solvent elimination and c) repeated solubilization/precipitation with petroleum ether/acetone. This crude extract was chromatographed on neutral alumina, which was exhaustively washed with chloroform before elution with chloroform:methanol, allowing the sequential separation of fatty acids and lecithin. Chromatographic behavior and mass spectra of the product are presented. This fast procedure yields fatty acid-free lecithin at a competitive cost.

An Environmental Friendly Procedure for Photometric Determination of Hypochlorite in Tap Water Employing a Miniaturized Multicommuted Flow Analysis Setup

A photometric procedure for the determination of ClO(-) in tap water employing a miniaturized multicommuted flow analysis setup and an LED-based photometer is described. The analytical procedure was implemented using leucocrystal violet (LCV; 4,4', 4 ''-methylidynetris (N, N-dimethylaniline), C(25)H(31)N(3)) as a chromogenic reagent. Solenoid micropumps employed for solutions propelling were assembled together with the photometer in order to compose a compact unit of small dimensions. After control variables optimization, the system was applied for the determination of ClO(-) in samples of tap water, and aiming accuracy assessment samples were also analyzed using an independent method. Applying the paired t-test between results obtained using both methods, no significant difference at the 95% confidence level was observed. Other useful features include low reagent consumption, 2.4 mu g of LCV per determination, a linear response ranging from 0.02 up to 2.0 mg L(-1) ClO(-), a relative standard deviation of 1.0% (n = 11) for samples containing 0.2 mg L(-1) ClO(-), a detection limit of 6.0 mu g L(-1) ClO(-), a sampling throughput of 84 determinations per hour, and a waste generation of 432 mu L per determination.

Bioassay guided purification of the antimicrobial fraction of a Brazilian propolis from Bahia state

Background: Brazilian propolis type 6 (Atlantic forest, Bahia) is distinct from the other types of propolis especially due to absence of flavonoids and presence of other non-polar, long chain compounds, but presenting good in vitro and in vivo antimicrobial activity. Several authors have suggested that fatty acids found in this propolis might be responsible for its antimicrobial activity; however, so far no evidence concerning this finding has been reported in the literature. The goals of this study were to evaluate the antibacterial activity of the main pure fatty acids in the ethanolic extract and fractions and elucidate the chemical nature of the bioactive compounds isolated from Brazilian propolis type 6. Methods: Brazilian propolis type 6 ethanolic extract (EEP), hexane fraction (H-Fr), major fatty acids, and isolated sub-fractions were analyzed using high performance liquid chromatography (HPLC), high resolution gas chromatography with flame ionization detection (HRGC-FID), and gas chromatography-mass spectrometry (GC-MS). Three sub-fractions of H-Fr were obtained through preparative HPLC. Antimicrobial activity of EEP, H-Fr, sub-fractions, and fatty acids were tested against Staphyloccus aureus ATCC 25923 and Streptococcus mutans Ingbritt 1600 using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results: EEP and H-Fr inhibited the growth of the microorganisms tested; nevertheless, no antimicrobial activity was found for the major fatty acids. The three sub-fractions (1, 2, and 3) were isolated from H-Fr by preparative HPLC and only sub-fraction 1 showed antimicrobial activity. Conclusion: a) The major fatty acids tested were not responsible for the antimicrobial activity of propolis type 6; b) Sub-fraction 1, belonging to the benzophenone class, was responsible for the antimicrobial activity observed in the present study. The identification of the bioactive compound will improve the development of more efficient uses of this natural product.

Extraction and purification of total RNA from banana tissues (small scale)

Introduction. This protocol aims at preparing total RNA for gene expression analysis by Northern blots, RT-PCR and real-time quantitative PCR; cDNA isolation by RTPCR; and cDNA library construction. The principle, key advantages, starting plant material, time required for obtaining total RNA and expected results are presented. Materials and methods. This part describes the required materials and the 27 steps necessary for preparing RNA from peel and pulp fruit tissue: preparation of plant tissue powder, preparation of the complete RNA extraction buffer and isolation of RNA from ground banana fruit tissue. Results. Extraction of total RNA by the method described makes it possible to achieve electrophoresis under denatured conditions and in vitro reverse transcription. An example for Northern blot analysis is illustrated.

Purification, crystallization and preliminary crystallographic analysis of the catalytic domain of the extracellular cellulase CBHI from Trichoderma harzianum

The filamentous fungus Trichoderma harzianum has a considerable cellulolytic activity that is mediated by a complex of enzymes which are essential for the hydrolysis of microcrystalline cellulose. These enzymes were produced by the induction of T. harzianum with microcrystalline cellulose (Avicel) under submerged fermentation in a bioreactor. The catalytic core domain (CCD) of cellobiohydrolase I (CBHI) was purified from the extracellular extracts and submitted to robotic crystallization. Diffraction-quality CBHI CCD crystals were grown and an X-ray diffraction data set was collected under cryogenic conditions using a synchrotron-radiation source.

Purification and characterization of an antimicrobial peptide produced by Pseudomonas sp strain 4B

An antimicrobial peptide produced by a bacterium isolated from the effluent pond of a bovine abattoir was purified and characterized. The strain was characterized by biochemical profiling and 16S rDNA sequencing as Pseudomonas sp. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A major band on SDS-PAGE suggested that the antimicrobial peptide has a molecular mass of about 30 kDa. The substance was inhibitory to a broad range of indicator strains, including pathogenic and food spoilage bacteria such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, among other. The partially purified antimicrobial substance remained active over a wide temperature range and was resistant to all proteases tested. This substance showed different properties than other antimicrobials from Pseudomonas species, suggesting a novel antimicrobial peptide was characterized.