A fast protocol for purification of translating mRNAs

We present a protocol that enables easy extraction of translating mRNAs by purification of tagged ribosomes and associated mRNAs. By studying three different examples of stress influenced genes, we demonstrate the effectiveness and compare our rapid method to an older protocol (Halbeisen et al., 2009).

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.

Application of OSN in membrane cascade for purification of the API Amoxicillin

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

High Throughput Purification of Combinatorial Libraries by Preparative LC/MS

Viime vuosien nopea kehitys on kiihdyttänyt uusien lääkkeiden kehittämisprosessia. Kombinatorinen kemia on tehnyt mahdolliseksi syntetisoida suuria kokoelmia rakenteeltaan toisistaan poikkeavia molekyylejä, nk. kombinatorisia kirjastoja, biologista seulontaa varten. Siinä molekyylien rakenteeseen liittyvä aktiivisuus tutkitaan useilla erilaisilla biologisilla testeillä mahdollisten "osumien" löytämiseksi, joista osasta saatetaan myöhemmin kehittää uusia lääkeaineita. Jotta biologisten tutkimusten tulokset olisivat luotettavia, on syntetisoitujen komponenttien oltava mahdollisimman puhtaita. Tämän vuoksi tarvitaan HTP-puhdistusta korkealaatuisten komponenttien ja luotettavan biologisen tiedon takaamiseksi. Jatkuvasti kasvavat tuotantovaatimukset ovat johtaneet näiden puhdistustekniikoiden automatisointiin ja rinnakkaistamiseen. Preparatiivinen LC/MS soveltuu kombinatoristen kirjastojen nopeaan ja tehokkaaseen puhdistamiseen. Monet tekijät, esimerkiksi erotuskolonnin ominaisuudet sekä virtausgradientti, vaikuttavat preparatiivisen LC/MS puhdistusprosessin tehokkuuteen. Nämä parametrit on optimoitava parhaan tuloksen saamiseksi. Tässä työssä tutkittiin emäksisiä komponentteja erilaisissa virtausolosuhteissa. Menetelmä kombinatoristen kirjastojen puhtaustason määrittämiseksi LC/MS-puhdistuksen jälkeen optimoitiin ja määritettiin puhtaus joillekin komponenteille eri kirjastoista ennen puhdistusta.

Fast and economic immobilization methods described for non-commercial Pseudomonas lipases

There is an increasing interest to seek new enzyme preparations for the development of new products derived from bioprocesses to obtain alternative bio-based materials. In this context, four non-commercial lipases from Pseudomonas species were prepared, immobilized on different low-cost supports, and examined for potential biotechnological applications. Results: To reduce costs of eventual scaling-up, the new lipases were obtained directly from crude cell extracts or from growth culture supernatants, and immobilized by simple adsorption on Accurel EP100, Accurel MP1000 and Celite (R) 545. The enzymes evaluated were LipA and LipC from Pseudomonas sp. 42A2, a thermostable mutant of LipC, and LipI. 3 from Pseudomonas CR611, which were produced in either homologous or heterologous hosts. Best immobilization results were obtained on Accurel EP100 for LipA and on Accurel MP1000 for LipC and its thermostable variant. Lip I. 3, requiring a refolding step, was poorly immobilized on all supports tested ( best results for Accurel MP1000). To test the behavior of immobilized lipases, they were assayed in triolein transesterification, where the best results were observed for lipases immobilized on Accurel MP1000. Conclusions: The suggested protocol does not require protein purification and uses crude enzymes immobilized by a fast adsorption technique on low-cost supports, which makes the method suitable for an eventual scaling up aimed at biotechnological applications. Therefore, a fast, simple and economic method for lipase preparation and immobilization has been set up. The low price of the supports tested and the simplicity of the procedure, skipping the tedious and expensive purification steps, will contribute to cost reduction in biotechnological lipase-catalyzed processes.

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.

Purification of Hydrocarbon Waste Streams

Purification of hydrocarbon waste streams is needed to recycle valuable hydrocarbon products, reduce hazardous impacts on environment, and save energy. To obtain these goals, research must be focused on the search of effective and feasible purification and re-refining technologies. Hydrocarbon waste streams can contain both deliberately added additives to original product and during operation cycle accumulated undesired contaminants. Compounds may have degenerated or cross-reacted. Thus, the presence of unknown species cause additional challenges for the purification process. Adsorption process is most suitable to reduce impurities to very low concentrations. Main advantages are availability of selective commercial adsorbents and the regeneration option to recycle used separation material. Used hydrocarbon fraction was purified with various separation materials in the experimental part. First screening of suitable materials was done. In the second stage, temperature dependence and adsorption kinetics were studied. Finally, one fixed bed experiment was done with the most suitable material. Additionally, FTIR-measurements of hydrocarbon samples were carried out to develop a model to monitor the concentrations of three target impurities based on spectral data. Adsorption capacities of the tested separation materials were observed to be low to achieve high enough removal efficiencies for target impurities. Based on the obtained data, batch process would be more suitable than a fixed bed process and operation at high temperatures is favorable. Additional pretreatment step is recommended to improve removal efficiency. The FTIR-measurement was proven to be a reliable and fast analysis method for challenging hydrocarbon samples.

The isolation, partial purification and preliminary characterization of a growth factor from chick embryo brains

Chicl( brain growth factor (CBGF) is a mitogen isolated from embryonic chick brains thought to have a potential role as a trophic factor involved in nerve dependent amphibian limb regeneration. In addition, CBGF stimulates 3H-thymidine incorporation in chick embryo brain astrocytes in vitro. In this study, cultured chick embryo brain non-neuronal cells were employed in a bioassay to monitor CBGF activity throughout various stages of its pllrification. Cell culture and assay conditions were optimized. Nonneuronal cells grew best on collagen-coated culture dishes in complete medium, were most responsive to a growth stimulus [10% fetal bovine serum (FBS)] at the second and third subcultures, and were healthiest when rendered "quiescent" in medium supplemented with 1% FBS. The most effective bioassay conditions consisted of a minimum 14.5 hour "quiescence" time (24 hours was used), a 6 hour "prestimulation" time, and a 24 hour 3H-thymidine labeling time. Four-day subconfluent primary non-neuronal cells consisted of 6.63% GFAP positive cells; as a result cultures were thought to be mainly composed of astroblasts. CBGF was purified from 18-day chick embryo brains by ultrafiltration through Amicon PM-30 and YM-2 membranes, size exclusion chromatography through a Biogel P6 column, and analytical reverse-phase high-performance liquid chromatography (rp-HPLC). The greatest activity resided in rp-HPLC fraction #7 (10 ng/ml) which was as effective as 10% FBS at stimulating 3H-thymidine incorporation in chick embryo brain nonneuronal cells. Although other researchers report the isolation of a mitogenic fraction consisting of 5'-GMP from the embryonic chick brain, UV absorbance spectra, rp-HPLC elution profiles, and fast atom bombardment (FAB) mass spectra indicated that CBGF is neither 5'-GMP nor 51-AMP. 2 Moreover, commercially available 5t-GMP was inhibitory to 3H-thymidine incorporation in the chick non-neuronal cells, while Sf-AMP had no effect. Upon treatment with pronase, the biological activity of fraction P6-3 increased; this increase was nearly 30% greater than what would be expected from a simple additive effect of any mitogenic activity of pronase alone together with P6-3 alone. This may suggest the presence of an inhibitor protein. The bioactive component may be a protein protected by a nucleoside/nucleotide or simply a nucleoside/nucleotide acting alone. While the FAB mass spectrum of rp-HPLC fraction #7 did not reveal molecular weight or sequence information, the ion of highest molecular weight was observed at m/z 1610; this is consistent with previous estimations of CBGF's size. 3

Purification par affinité et marquage isotopique spécifique pour études d’ARN fonctionnels

Il existe un lien étroit entre la structure tridimensionnelle et la fonction cellulaire de l’ARN. Il est donc essentiel d’effectuer des études structurales de molécules d’ARN telles que les riborégulateurs afin de mieux caractériser leurs mécanismes d’action. Une technique de choix, permettant d’obtenir de l’information structurale sur les molécules d’ARN est la spectroscopie RMN. Cette technique est toutefois limitée par deux difficultés majeures. Premièrement, la préparation d’une quantité d’ARN nécessaire à ce type d’étude est un processus long et ardu. Afin de résoudre ce problème, notre laboratoire a développé une technique rapide de purification des ARN par affinité, utilisant une étiquette ARiBo. La deuxième difficulté provient du grand recouvrement des signaux présents sur les spectres RMN de molécules d’ARN. Ce recouvrement est proportionnel à la taille de la molécule étudiée, rendant la détermination de structures d’ARN de plus de 15 kDa extrêmement complexe. La solution émergeante à ce problème est le marquage isotopique spécifique des ARN. Cependant, les protocoles élaborées jusqu’à maintenant sont très coûteux, requièrent plusieurs semaines de manipulation en laboratoire et procurent de faibles rendements. Ce mémoire présente une nouvelle stratégie de marquage isotopique spécifique d’ARN fonctionnels basée sur la purification par affinité ARiBo. Cette approche comprend la séparation et la purification de nucléotides marqués, une ligation enzymatique sur support solide, ainsi que la purification d’ARN par affinité sans restriction de séquence. La nouvelle stratégie développée permet un marquage isotopique rapide et efficace d’ARN fonctionnels et devrait faciliter la détermination de structures d’ARN de grandes tailles par spectroscopie RMN.

Adaptations de la méthode de purification d’ARN par affinité avec l’étiquette ARiBo

Dans les dernières années, une explosion de la recherche sur les ARN a eu lieue à cause de nombreuses découvertes démontrant l’importance de l’ARN dans plusieurs processus biologiques. Ainsi, de grandes quantités d’ARN sont devenues indispensables au bon déroulement de plusieurs études, notamment pour la biologie structurale et la caractérisation fonctionnelle. Cependant, il existe encore peu de méthodes de purification simples, efficaces, fiables et produisant un ARN sous forme native. Dans les dernières années, le laboratoire Legault a mis au point une méthode de purification par affinité utilisant une étiquette ARiBo pour la purification d’ARN transcrits in vitro par la polymérase à ARN du phage T7. Cette méthode de purification d’ARN a été spécifiquement développée pour maximiser la pureté et le rendement. De plus, elle est très rapide et fonctionne avec plusieurs types d’ARN. Cependant, comme plusieurs autres méthodes de purification, cette méthode produit des ARN avec des extrémités 5′ hétérogènes. Dans ce mémoire, des solutions sont proposées pour remédier au problème d’hétérogénéité en 5ʹ′ des ARN transcrits avec la polymérase à ARN du phage T7 et purifiés par la méthode ARiBo. La première solution consiste à choisir la séquence en 5′ parmi celles des 32 séquences testées qui ne présentent pas d’hétérogénéité en 5ʹ′. La seconde solution est d’utiliser une étiquette clivable en 5ʹ′ de l’ARN d’intérêt, tel que le ribozyme hammerhead, déjà utilisée pour ce genre d’application, ou le système CRISPR/Cse3 que nous proposons dans l’article présenté dans ce mémoire. De plus, nous avons adapté la méthode ARiBo pour rendre possible la purification d’un long ARN de 614 nt, le polycistron miR-106b-25. Nous avons également démontré la possibilité d’utiliser la méthode ARiBo pour l’isolation de protéines qui se lient à un ARN donné, le précurseur de miRNA pre-miR-153-2. En conclusion, ce mémoire démontre la possibilité d’adapter la méthode ARiBo à plusieurs applications.

Cloning, overexpression, and purification of functional human purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for human PNP causes T-cell deficiency as the major physiological defect. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant tissue rejection, psoriasis, rheumatoid arthritis, lupus, and T-cell lymphomas. Human PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation. In addition, bacterial PNP has been used as reactant in a fast and sensitive spectrophotometric method that allows both quantitation of inorganic phosphate (Pi) and continuous assay of reactions that generate P i such as those catalyzed by ATPases and GTPases. Human PNP may therefore be an important biotechnological tool for P i detection. However, low expression of human PNP in bacterial hosts, protein purification protocols involving many steps, and low protein yields represent technical obstacles to be overcome if human PNP is to be used in either high-throughput drug screening or as a reagent in an affordable P i detection method. Here, we describe PCR amplification of human PNP from a liver cDNA library, cloning, expression in Escherichia coli host, purification, and activity measurement of homogeneous enzyme. Human PNP represented approximately 42% of total soluble cell proteins with no induction being necessary to express the target protein. Enzyme activity measurements demonstrated a 707-fold increase in specific activity of cloned human PNP as compared to control. Purification of cloned human PNP was achieved by a two-step purification protocol, yielding 48 mg homogeneous enzyme from 1 L cell culture, with a specific activity value of 80 U mg -1. © 2002 Elsevier Science (USA). All rights reserved.

Adaptabilidade diferencial das variantes moleculares slow e fast da esterase-5 de Drosophila mulleri

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Lignases Production and Partial Purification of Mnp from Brazilian Fungal Isolate in Submerged Fermentation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Purification of coagulation factor VIII using chromatographic methods. Direct chromatography of plasma in anion exchange resins

Coagulation factor VIII (FVIII) concentrates are used in the treatment of patients with Hemophilia A. Human FVIII was purified directly from plasma using anion exchange chromatography followed by gel filtration. Three Q-Sepharose resins were tested, resulting in 40% recovery of FVIII activity using Q-Sepharose XL resin, about 80% using Q-Sepharose Fast Flow and 70% using the Q-Sepharose Big Beads. The vitamin K-dependent coagulation factors co-eluted with FVIII from the anion exchange columns. In the second step of purification, when Sepharose 6FF was used, 70% of FVIII activity was recovered free from vitamin K-dependent factors.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.