1,721,033 research outputs found
Online Enantioselective Capillary Electrophoretic Method for Screening Cytochrome P450 3A4 Inhibitors.
No full textThe market share of single-enantiomer drugs is steadily increasing. The pharmacodynamics and pharmacokinetics of individual enantiomers can differ considerably. Thus, their characteristics have to be addressed as early as possible in the development process of new pharmaceuticals. Capillary electrophoresis is a promising technique for enantioselective drug metabolism studies due to highly effective separations, minuscule consumption of sample and reagents, compatibility with a variety of detection techniques, high-throughput via automation, and the implementation of online procedures. An online method comprised of the diffusion-based mixing of cytochrome P450 3A4 with racemic ketamine, incubation of the enzyme reaction, separation of the reaction products S- and R-norketamine, and their quantification is presented in this chapter. Since diffusion is an inherent property of all molecules, the method enables the addition of virtually any compound to the reaction mixture without the need for additional optimization of the mixing conditions, and thus can be favorably used for the rapid screening of putative cytochrome P450 3A4 inhibitors
Electrophoretic mobility shift assays with GFP-tagged proteins (GFP-EMSA)
Full text linkThe electrophoretic mobility shift assay (EMSA) is commonly used for the study of nucleic acid-binding proteins. The technique can be used to demonstrate that a protein is binding to RNA or DNA through visualization of a shift in electrophoretic mobility of the nucleic acid band. A major disadvantage of the EMSA is that it does not always provide an absolute certitude that the band shift is due to the protein under scrutiny, as contaminants in the sample could also cause the band shift. Here we describe a variation of the standard EMSA allowing to visualize with added certitude, the co-localized band shifts of a GFP-tagged protein binding to its cognate nucleic acid target sequence stained with an intercalator, such as GelRed. Herein, we present an illustrative protocol of this useful technique called GFP-EMSA along with specific notes on its advantages and limitations
Enzymatic Bioautographic Methods
No full textEnzymatic bioautography enables the detection of enzyme inhibitors absorbed on a thin-layer chromatography plate. Therefore, it is an assay format that is particularly useful for the detection of inhibitors present in complex mixtures. The inhibition properties of compounds separated by thin-layer chromatography can be directly analyzed to produce an inhibition profile. Here, we describe the conditions to detect inhibitor of the enzymes xanthine oxidase and β-glucosidase immobilized on agar gel.Fil: Ramallo, Ivana Ayelen. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Orgánica. Área Farmacognosia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Salazar, Mario Oscar. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Orgánica. Área Farmacognosia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Furlan, Ricardo Luis Eugenio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Orgánica. Área Farmacognosia; Argentin
High-throughput differential scanning fluorimetry of GFP-tagged proteins
Full text linkDifferential scanning fluorimetry is useful for a wide variety of applications including characterization of protein function, structure–activity relationships, drug screening, and optimization of buffer conditions for protein purification, enzyme activity, and crystallization. A limitation of classic differential scanning fluorimetry is its reliance on highly purified protein samples. This limitation is overcome through differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP). DSF-GTP specifically measures the unfolding and aggregation of a target protein fused to GFP through its proximal perturbation effects on GFP fluorescence. As a result of this unique principle, DSF-GTP can specifically measure the thermal stability of a target protein in the presence of other proteins. Additionally, the GFP provides a unique in-assay quality control measure. Here, we describe the workflow, steps, and important considerations for executing a DSF-GTP experiment in a 96-well plate format
Peptide affinity chromatography based on combinatorial strategies for protein purification
No full textWe describe a method to develop affinity chromatography matrices with short peptide ligands for protein purification. The method entitles the following: a) synthesis of a combinatorial library on the HMBA-ChemMatrix resin by the divide-couple-recombine (DCR) method using the Fmoc chemistry, b) library screening with the protein of interest labeled with a fluorescent dye or biotin, c) identification of peptides contained on positive beads by tandem matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS), d) solid phase peptide ligands synthesis and immobilization in chromatographic supports, e) evaluation of protein adsorption on peptide affinity matrices from the equilibrium isotherms and breakthrough curves.Fil: Camperi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; ArgentinaFil: Martínez Ceron, María Camila. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; ArgentinaFil: Giudicessi, Silvana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; ArgentinaFil: Marani, Mariela Mirta. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; ArgentinaFil: Albericio Palomera, Fernando. Universidad de Barcelona; EspañaFil: Cascone, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; Argentin
Design of affinity chromatography peptide ligands through combinatorial peptide libraries screening
No full textIn this chapter, a protocol to design affinity chromatography matrices with short peptide ligands immobilized for protein purification is described. The first step consists of the synthesis of a combinatorial peptide library on the hydroxymethylbenzoyl (HMBA)-ChemMatrix resin by the divide–couple–recombine (DCR) method using the Fmoc chemistry. Next, the library is screened with the protein of interest labeled with a fluorescent dye or biotin. Subsequently, peptides contained on positive beads are identified by tandem matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS), and those sequences showing greater consensus are synthesized in larger quantities and immobilized on chromatographic supports. Finally, target protein adsorption on peptide affinity matrices is evaluated through equilibrium adsorption isotherms and breakthrough curves.Fil: Barredo, Gabriela Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Saavedra, Soledad Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Martínez Ceron, María Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Giudicessi, Silvana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Marani, Mariela Mirta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto Patagónico para el Estudio de los Ecosistemas Continentales; Argentin
Challenges in dairy whey protein purification
No full textCheese whey, a by-product generated during the manufacture of cheese, with an annual world production of almost 200 million tons, is currently considered a waste for many cheese-making companies. However, many valuable and marketable proteins originally contained in the milk remain in the whey and their isolation from this source would increase the added value of whey. Due to their complex nature, the isolation of cheese whey proteins represents an enormous challenge, which increases when considering an industrial scale. Moreover, when high volumes of cheese whey -or even milk- need to be processed, most of the protein purification models usually used in the biopharma/biotechnology industries fail to be applied. This chapter will discuss some novel and scalable approaches for the processing of cheese whey for protein production, with potential application at industrial scale.Fil: Urtasun, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Baieli, María Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Hirsch, Daniela Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Miranda, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Cascone, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Wolman, Federico Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentin
Molecular and biochemical characterization of the parvulin-type PPIases in Lotus japonicus
Full text linkThe cis/trans isomerization of the peptide bond preceding proline is an intrinsically slow process, although important in many biological processes in both prokaryotes and eukaryotes. In vivo, this isomerization is catalyzed by peptidyl-prolyl cis/trans-isomerases (PPIases). Here, we present the molecular and biochemical characterization of parvulin-type PPIase family members of the model legume Lotus japonicus, annotated as LjPar1, LjPar2, and LjPar3. Although LjPar1 and LjPar2 were found to be homologous to PIN1 (Protein Interacting with NIMA)-type parvulins and hPar14 from human, respectively, LjPar3 represents a novel multidomain parvulin, apparently present only in plants, that contains an active carboxyl-terminal sulfurtransferase domain. All Lotus parvulins were heterologously expressed and purified from Escherichia coli, and purified protein verification measurements used a liquid chromatography-mass spectrometry-based proteomic method. The biochemical characterization of the recombinant Lotus parvulins revealed that they possess PPIase activity toward synthetic tetrapeptides, although they exhibited different substrate specificities depending on the amino acid amino terminal to proline. These differences were also studied in a structural context using molecular modeling of the encoded polypeptides. Real-time reverse transcription-polymerase chain reaction revealed that the three parvulin genes of Lotus are ubiquitously expressed in all plant organs. LjPar1 was found to be up-regulated during the later stages of nodule development. Subcellular localization of LjPar-enhanced Yellow Fluorescence Protein (eYFP) fusions expressed in Arabidopsis (Arabidopsis thaliana) leaf epidermal cells revealed that LjPar1- and LjPar2-eYFP fusions were localized in the cytoplasm and in the nucleus, in contrast to LjPar3-eYFP, which was clearly localized in plastids. Divergent substrate specificities, expression profiles, and subcellular localization indicate that plant parvulin-type PPIases are probably involved in a wide range of biochemical and physiological processes
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