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 b
- PDF / 1,782,893 Bytes
- 17 Pages / 504 x 720 pts Page_size
- 94 Downloads / 146 Views
1. Introduction Structural studies of membrane proteins require large amounts of purified and concentrated proteins (1). However, most membrane proteins are not naturally abundant and overexpression is one way that has been developed to overcome such difficulty. Bacteria, such as Escherichia coli (E. coli) cells, are the preferred host for recombinant protein expression because they are rather easy to genetically manipulate and expression is fast, typically producing protein in a single day or less than 24 h. A large number of vectors with different fusion tags for purification have been developed over the last decades (2). Histidine fusion tag is one of
Jean-Jacques Lacapère (ed.), Membrane Protein Structure Determination: Methods and Protocols, Methods in Molecular Biology, vol. 654, DOI 10.1007/978-1-60761-762-4_3, © Springer Science+Business Media, LLC 2010
29
30
Robert and Lacapère
the most commonly used since it often enables easy purification on NTA-Ni affinity columns. TranSlocator PrOtein (TSPO), previously named peripheraltype benzodiazepines receptor (PBR) is a transmembrane protein mostly located in mitochondria and initially discovered as a class of binding site for benzodiazepine distinct from the GABAa receptors from the central nervous system (3). TSPO expression is too low to permit easy purification from native membranes. cDNA from mouse was cloned, inserted into vectors, and E. coli cells were transformed with these vectors (4, 5). A small fraction of TSPO was found in bacterial membrane, and the major fraction was detected in inclusion bodies. Different fusion tags for purification were tested (4, 5), good results were obtained with six histidines fusion tag added in N-terminal position of the TSPO. Theoretically, the fusion tag placed in C-terminal position permits to remove non-fully expressed proteins, but in the case of TSPO, the C-terminal domain plays an important role in cholesterol binding (6) and transport (5) and thus is affected by the presence of a tag. The present chapter describes production and purification of recombinant TSPO (RecTSPO). The first part is a comparison of RecTSPO production in an incubator with Luria–Bertani (LB) broth or minimum medium (M9) complemented with isotopes for nuclear magnetic resonance (NMR) studies and in a fermentor with LB broth. The second part describes purification on affinity column. We developed two protocols with Ni-NTA resin, large-scale purification with fast protein liquid chromatography (FPLC) procedure and small-scale purification with “manual chromatography”. Advantages and disadvantages are discussed in the light of the target, that is, the structural approach used. Exchange of detergent on Ni-NTA resin is presented, since deuterated detergents are needed for NMR studies, whereas nonionic detergents are requested for mass spectroscopy analysis with MALDITOF. The last part presents protocols for characterisation of detergent-purified RecTSPO, for instance, to find optimal conditions of detergent/protein ratios, which appeared very u
Data Loading...
