Bacterial overexpressed membrane proteins: an example: the TSPO.
The mitochondrial membrane TranSlocator PrOtein (TSPO) is a 18-kDa transmembrane protein involved in various mitochondrial functions, among which the best characterised is cholesterol transport and steroid formation. Determination of its structure would be an important step to understand the mechanism of transport and its regulation. Purification from native membranes is difficult in respect with amounts of homogeneous purified proteins needed for biophysical, structural, and functional studies. Efficient heterologous overexpression in bacterial system, purification on affinity column, and biochemical characterisation has been successfully developed. Large-scale production of detergent-solubilized TSPO has been obtained with fermentation coupled to fast protein liquid chromatography procedure. Small-scale production at lower cost for isotopically labelled recombinant TSPO and/or detergent is also presented.
Robert
Jean-Claude
JC
INSERM U773, Centre de Recherche Biomédicale Bichat-Beaujon (CRB3), Faculté de Médecine X, Bichat, Université Paris 7, Paris, France.
Lacapère
Jean-Jacques
JJ
D000818:Animals; D019149:Bioreactors; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D018079:Receptors, GABA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
C463819:Bzrp protein, mouse; D003902:Detergents; D018079:Receptors, GABA; C499229:TSPO protein, human