Native membrane proteins vs. yeast recombinant: an example: the mitochondrial ADP/ATP carrier

Autor: Arnou, B., Dahout-Gonzalez, C., Pelosi, L., Lauquin, G. J., Brandolin, G., Véronique Trézéguet
Přispěvatelé: Institut de biochimie et génétique cellulaires (IBGC), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Biochimie et biophysique des systèmes intégrés (BBSI), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de biochimie et génétique cellulaires ( IBGC ), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique ( CNRS ), Biochimie et biophysique des systèmes intégrés ( BBSI ), Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ), Grellety, Marie-Lise
Jazyk: angličtina
Rok vydání: 2010
Předmět:
Zdroj: Methods in Molecular Biology
Methods in Molecular Biology, Humana Press/Springer Imprint, 2010, 654, pp.19-28
Methods in Molecular Biology (Clifton then Totova)
Methods in Molecular Biology (Clifton then Totova), Humana Press (Springer Imprint), 2010, 654, pp.19-28
HAL
Methods in Molecular Biology, 2010, 654, pp.19-28
ISSN: 1064-3745
1940-6029
Popis: The mitochondrial ADP/ATP carrier (Ancp) has long been a paradigm for studies of the mitochondrial carrier family due to, among other properties, its natural abundance and the existence of specific inhibitors, namely, carboxyatractyloside (CATR) and bongkrekic acid (BA), which lock the carrier under distinct and stable conformations. Bovine Anc1p isolated in complex with CATR in the presence of an aminoxyde detergent (LAPAO) was crystallized and its 3D structure determined. It is the first mitochondrial carrier structure resolved at high resolution (2.2 A, as reported by Pebay-Peyroula et al. (Nature 426:39-44, 2003)). Analyses revealed a monomer while most of the biochemical studies led to hypothesize Ancp functions as a dimer. To address the structural organization issue, we engineered a mutant of the yeast Ancp that corresponds to a covalent homodimer in view of 3D structure determination. We compare in this chapter the purification yield and quality of the chimera tagged either with six histidines at its C-ter end or nine histidines at its N-ter. We show that, as expected, length and position of the tag are important criteria for qualitative purification. We also discuss the advantages and drawbacks of purifying Ancp either from a natural source or from engineered yeast cells.
Databáze: OpenAIRE
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