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1.
Protein Expr Purif ; 64(1): 1-7, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18835448

RESUMO

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. Structure determination of G protein-coupled receptors and other applications require milligram quantities of purified receptor proteins on a regular basis. Recombinant GPCRs fused to a heterologous biotinylation domain were produced in the yeast Pichia pastoris. We describe an efficient method for their rapid purification that relies on the capture of these receptors with streptavidin immobilized on agarose beads, and their subsequent release by enzymatic digestion with TEV protease. This method has been applied to several GPCRs belonging to the class A rhodopsin subfamily, leading to high yields of purified proteins; it represents a method of choice for biochemical and biophysical studies when large quantities of purified GPCRs are needed.


Assuntos
Receptores Acoplados a Proteínas G/isolamento & purificação , Biotinilação , Endopeptidases/metabolismo , Humanos , Microesferas , Modelos Biológicos , Pichia/genética , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Sefarose/metabolismo , Solubilidade , Estreptavidina/isolamento & purificação , Estreptavidina/metabolismo
2.
Protein Expr Purif ; 60(2): 214-20, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18522870

RESUMO

A method is presented to produce large amounts of Bcl-2 and Bcl-x(L), two anti-apoptotic proteins of considerable biomedical interest. Expression constructs were prepared in which the Escherichia coli protein TolAIII, known to promote over expression of soluble product, was added to the N-terminus of Bcl-2 or Bcl-x(L) proteins, which had their C-terminal hydrophobic anchors deleted. Here the expression of these TolAIII-fusion constructs, followed by a two-step metal-affinity based purification protocol is described. The method delivers at least 20 and 10mg of more than 90% pure TolAIII-Bcl-x(L)DeltaC and TolAIII-Bcl-2(2)DeltaC proteins, respectively, per liter of E. coli cell culture. The proteins are released by proteolysis with thrombin providing > 12 mg of Bcl-x(L)DeltaC or > 6 mg of Bcl-2(2)DeltaC per liter of E. coli cell culture with a purity of more than 95%. Whereas Bcl-x(L)DeltaC is soluble both before and after TolAIII removal, Triton X-100 can significantly increase the extraction of TolAIII- Bcl-2(2)DeltaC from the bacterial cells and its subsequent solubility. Far-UV CD spectroscopy demonstrated that they both have an alpha-helical structure. Fluorescence spectroscopy was used to quantitatively analyze the binding of the respiratory inhibitor antimycin A to recombinant Bcl-2 and Bcl-x(L) proteins as well as the displacement of this ligand from the hydrophobic pocket with BH3 Bad-derived peptide. Purified Bcl-x(L)DeltaC and Bcl-2(2)DeltaC both protect isolated mitochondria from Bax-induced release of cytochrome c. The ensemble of data shows that the expressed proteins are correctly folded and functional. Therefore, the TolAIII-fusion system provides a convenient tool for functional characterization and structural studies of anti-apoptotic proteins.


Assuntos
Proteínas de Escherichia coli/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes de Fusão/genética , Proteína bcl-X/genética , Animais , Sequência de Bases , Dicroísmo Circular , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Proteínas de Escherichia coli/isolamento & purificação , Mitocôndrias Hepáticas/enzimologia , Plasmídeos , Proteínas Proto-Oncogênicas c-bcl-2/isolamento & purificação , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/isolamento & purificação , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Proteína bcl-X/isolamento & purificação
3.
Protein Sci ; 15(5): 1115-26, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16597836

RESUMO

We have optimized the expression level of 20 mammalian G protein-coupled receptors (GPCRs) in the methylotrophic yeast Pichia pastoris. We found that altering expression parameters, including growth temperature, and supplementation of the culture medium with specific GPCR ligands, histidine, and DMSO increased the amount of functional receptor, as assessed by ligand binding, by more than eightfold over standard expression conditions. Unexpectedly, we found that the overall amount of GPCR proteins expressed, in most cases, varied only marginally between standard and optimized expression conditions. Accordingly, the optimized expression conditions resulted in a marked fractional increase in the ratio of ligand binding-competent receptor to total expressed receptor. The results of this study suggest a general approach for increasing yields of functional mammalian GPCRs severalfold over standard expression conditions by using a set of optimized expression condition parameters that we have characterized for the Pichia expression system. Overall, we have more than doubled the number of GPCR targets that can be produced in our laboratories in sufficient amounts for structural studies.


Assuntos
Clonagem Molecular/métodos , Pichia/química , Receptores Acoplados a Proteínas G/química , Sequência de Aminoácidos , Proteínas de Transporte , Expressão Gênica , Immunoblotting , Cinética , Ligantes , Dados de Sequência Molecular , Ligação Proteica , Ensaio Radioligante , Receptores Acoplados a Proteínas G/análise , Receptores Acoplados a Proteínas G/isolamento & purificação , Técnicas de Cultura de Tecidos
4.
Curr Opin Pharmacol ; 9(5): 629-35, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19443270

RESUMO

G-protein-coupled receptors (GPCRs), the largest family of membrane proteins, represent ideal therapeutic targets for a number of disorders and diseases. Besides cell-based assays and high throughput screening (HTS), and thanks to the availability of milligram quantities of active purified receptors, protein-based approaches focusing on soluble GPCRs are growingly being used in this drug discovery effort. Along with the exploitation of GPCRs structures, innovative biochemical and biophysical approaches open up new routes for improving the knowledge of structure-activity relationships, for the identification of novel interacting partners and for the determination of receptor behaviour in different model environments. This review summarizes the state-of-the-art methodologies that robustly allow for the production and purification of soluble and active GPCRs, as well as the main outcomes that have been recently gained in GPCR biology using a panel of such protein-based approaches.


Assuntos
Desenho de Fármacos , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Proteômica , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Animais , Sítios de Ligação , Humanos , Ligantes , Estrutura Molecular , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/isolamento & purificação , Relação Estrutura-Atividade
5.
J Struct Funct Genomics ; 7(2): 77-91, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17120110

RESUMO

Production of recombinant receptors has been one of the major bottlenecks in structural biology on G protein-coupled receptors (GPCRs). The MePNet (Membrane Protein Network) was established to overexpress a large number of GPCRs in three major expression systems, based on Escherichia coli, Pichia pastoris and Semliki Forest virus (SFV) vectors. Evaluation by immunodetection demonstrated that 50% of a total of 103 GPCRs were expressed in bacterial inclusion bodies, 94% in yeast cell membranes and 95% in SFV-infected mammalian cells. The expression levels varied from low to high and the various GPCR families and subtypes were analyzed for their expressability in each expression system. More than 60% of the GPCRs were expressed at milligram levels or higher in one or several systems, compatible to structural biology applications. Functional activity was determined by binding assays in yeast and mammalian cells and the correlation between immunodetection and binding activity was analyzed.


Assuntos
Escherichia coli , Expressão Gênica , Genômica , Proteínas de Membrana , Pichia , Receptores Acoplados a Proteínas G , Vírus da Floresta de Semliki , Animais , Western Blotting , Clonagem Molecular , Escherichia coli/genética , Genes Bacterianos , Genes Fúngicos , Genes Virais , Vetores Genéticos , Humanos , Immunoblotting , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pichia/genética , Ligação Proteica , Ensaio Radioligante , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Vírus da Floresta de Semliki/genética
6.
Glycobiology ; 13(3): 169-77, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12626404

RESUMO

Glycosylphosphatidylinositol (GPI)-anchored proteins have been identified in all eukaryotes. In fungi, structural and biosynthetic studies of GPIs have been restricted to the yeast Saccharomyces cerevisiae. In this article, four GPI-anchored proteins were purified from a membrane preparation of the human filamentous fungal pathogen Aspergillus fumigatus. Using new methodology applied to western blot protein bands, the GPI structures were characterized by ES-MS, fluorescence labeling, HPLC, and specific enzymatic digestions. The phosphatidylinositol moiety of the A. fumigatus GPI membrane anchors was shown to be an inositol-phosphoceramide containing mainly phytosphingosine and monohydroxylated C24:0 fatty acid. In constrast to yeast, only ceramide was found in the GPI anchor structures of A. fumigatus, even for Gel1p, a homolog of Gas1p in S. cerevisiae that contains diacylglycerol. The A. fumigatus GPI glycan moiety is mainly a linear pentomannose structure linked to a glucosamine residue: Manalpha1-3Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4GlcN.


Assuntos
Aspergillus fumigatus/química , Proteínas Fúngicas/química , Glicosilfosfatidilinositóis/química , Proteínas de Membrana/química , Western Blotting , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/química
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