Abstract
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.
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References
Lacapere J-J, Pebay-Peyroula E, Neumann J-M, Etchebest C (2007) Determining membrane protein structures: still a challenge! Trends Biochem Sci 32:259–270
Peti W, Page R (2007) Strategies to maximise heterologous protein expression in Escherichia coli with minimal cost. Protein Expr Purif 51:1–10
Papadopoulos V, Baraldi M, Guilarte TR, Knudsen TB, Lacapere J-J, Lindemann P, Noremberg MD, Nutt D, Weizman A, Zhang M-R, Gavish M (2006) Translocator protein (18 kDa): New nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function. Trends Pharmacol Sci 27:402–409
Garnier M, Dimchev AB, Boujrad N, Price JM, Musto NA, Papadopoulos V (1994) In vitro reconstitution of a functional peripheral-type benzodiazepine receptor from mouse Leydig tumor cells. Mol Pharmacol 5:201–211
Li H, Papadopoulos V (1998) Peripheric-type benzodiazepine receptor function in cholesterol transport. Identification of a putative cholesterol recognition/interaction aminoacid sequence and consensus pattern. Endocrynology 139:4991–4997
Jamin N, Neumann J-M, Ostuni MA, Kim NV, Yao ZX, Murail S, Robert J-C, Giatzakis C, Papadopoulos V, Lacapere J-J (2005) Characterization of the cholesterol recognition amino acid consensus sequence of the peripheral-type benzodiazepine receptor. Mol Endocrinol 19:588–594
Sambrook J, Fritsch EF, Maniatis M (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A (2003) ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31:3784–3788
Marley J, Lu M, Bracken C (2001) A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR 20:71–75
Charbonnier F, Köhler T, Pechère JC, Ducruix A (2001) Overexpression, refolding, and purification of the Histidine-tagged outer membrane efflux protein OprM of Pseudomonas aeruginosa. Protein Expr Purif 23:121–127
Baneres J-L, Martin A, Hullot P, Girard J-P, Rossi J-C, Parello J (2003) Structure-based analysis of GPCR function: conformational adaptation of both agonist and receptor upon leukotrienes B4 binding to recombinant BLT1. J Mol Biol 329:801–814
Bane SE, Velasquez JE, Robinson AK (2007) Expression and purification of milligram level of inactive G-protein coupled receptors in E. Coli. Protein Expr Purif 52:348–355
Lacapere J-J, Delavoie F, Li H, Péranzi G, Maccario J, Papadopulos V, Vidic B (2001) Structural and fonctional study of reconstituted peripheral benzodiazepine receptor. Biochem Biophys Res Commun 284:536–541
Columbus L, Lipfert J, Klock H, Millett I, Doniach S, Lesley SA (2006) Expression, purification, and characterization of Thermotoga maritima membrane proteins for structure determination. Protein Sci 15:1–15
Laemli UK (1970) Cleavage of structural proteins during the assembly of the head of acteriophage T4. Nature 227:680–685
Yan JX, Wait R, Berkelman T, Harry RA, Westbrook JA, Wheeler CH, Dunn MJ (2000) A modified silver staining protocol for visualisation of proteins compatible with matrix-assisted laser desorption/ionizatioin and electrospray ionization-mass spectroscopy. Electrophoresis 21:3666–3672
Riesenberg D, Gunthke R (1999) High cell density cultivation of microorganisms. Appl Microbiol Biotechnol 51:422–430
Acknowledgment
The authors would like to thank Professor V. Papadopoulos for the generous gift of TSPO plasmids. They would gratefully thank M.A. Ostuni for his help in the critical reading of this chapter. This work was supported by CNRS (Centre National de la Recherche Scientifique) and ANR (Agence National pour la Recherche) Grant 06-Blan-0190-01 to JJL.
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Robert, JC., Lacapère, JJ. (2010). Bacterial Overexpressed Membrane Proteins: An Example: The TSPO. In: Lacapère, JJ. (eds) Membrane Protein Structure Determination. Methods in Molecular Biology, vol 654. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-762-4_3
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DOI: https://doi.org/10.1007/978-1-60761-762-4_3
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