Native membrane proteins vs. yeast recombinant: an example: the mitochondrial ADP/ATP carrier.
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.
Arnou
Bertrand
B
Laboratoire de Physiologie Moléculaire et Cellulaire, Institut de Biochimie et Génétique Cellulaires-UMR 5095, CNRS-Université Bordeaux 2, Bordeaux, France.
Dahout-Gonzalez
Cécile
C
Pelosi
Ludovic
L
Lauquin
Guy J-M
GJ
Brandolin
Gérard
G
Trézéguet
Véronique
V
D000818:Animals; D001278:Atractyloside / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D001865:Bongkrekic Acid / Q000737:chemistry / Q000494:pharmacology; D002417:Cattle; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D011994:Recombinant Proteins; D001865:Bongkrekic Acid; D001278:Atractyloside; D000226:Mitochondrial ADP, ATP Translocases; C003853:carboxyatractyloside