Cell-Free Expression for the Study of Hydrophobic Proteins: The Example of Yeast ATP-Synthase Subunits.

Autor:	Larrieu I; SysTEMM, CNRS, UMR5095, IBGC, CS 61390, 1 rue Camille Saint-Saëns, 33077, Bordeaux, France.; Université de Bordeaux, Campus Carreire, 146 Rue Léo Saignat, 33000, Bordeaux, France., Tolchard J; CNRS, UMR 5248, CBMN, 33600, Pessac, France.; Université de Bordeaux, Campus de Pessac, Allée Geoffroy Saint-Hilaire, 33600, Pessac, France., Sanchez C; SysTEMM, CNRS, UMR5095, IBGC, CS 61390, 1 rue Camille Saint-Saëns, 33077, Bordeaux, France.; Université de Bordeaux, Campus Carreire, 146 Rue Léo Saignat, 33000, Bordeaux, France., Kone EY; SysTEMM, CNRS, UMR5095, IBGC, CS 61390, 1 rue Camille Saint-Saëns, 33077, Bordeaux, France.; Université de Bordeaux, Campus Carreire, 146 Rue Léo Saignat, 33000, Bordeaux, France., Barras A; SysTEMM, CNRS, UMR5095, IBGC, CS 61390, 1 rue Camille Saint-Saëns, 33077, Bordeaux, France.; CNRS, UMR 5248, CBMN, 33600, Pessac, France.; Université de Bordeaux, Campus de Pessac, Allée Geoffroy Saint-Hilaire, 33600, Pessac, France., Stines-Chaumeil C; CNRS, CRPP, UPR 8641, F-33600, Pessac, France.; Université de Bordeaux, CRPP, UPR 8641, F-33600, Pessac, France., Odaert B; CNRS, UMR 5248, CBMN, 33600, Pessac, France.; Université de Bordeaux, Campus de Pessac, Allée Geoffroy Saint-Hilaire, 33600, Pessac, France., Giraud MF; SysTEMM, CNRS, UMR5095, IBGC, CS 61390, 1 rue Camille Saint-Saëns, 33077, Bordeaux, France. marie-france.giraud@ibgc.cnrs.fr.; Université de Bordeaux, Campus Carreire, 146 Rue Léo Saignat, 33000, Bordeaux, France. marie-france.giraud@ibgc.cnrs.fr.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2017; Vol. 1635, pp. 57-90.
DOI:	10.1007/978-1-4939-7151-0_4
Abstrakt:	Small hydrophobic membrane proteins or proteins with hydrophobic domains are often difficult to produce in bacteria. The cell-free expression system was found to be a very good alternative for the expression of small hydrophobic subunits of the yeast ATP-synthase, such as subunits e, g, k, i, f and the membrane domain of subunit 4, proteins that are suspected to play a role in the stability of ATP-synthase dimers. All of these proteins could be produced in milligrams amounts using the cell-free "precipitate mode" and were successfully solubilized in the presence of lysolipid 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-1'-rac-glycerol. Purified proteins were also found suitable for structural investigations. An example is given with the NMR backbone assignment of the isotopically labeled subunit g. Protocols are also described for raising specific polyclonal antibodies against overexpressed cell-free proteins.
Databáze:	MEDLINE
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