Bcl-XL as a fusion protein for the high-level expression of membrane-associated proteins |
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Authors: | Thai Khang Choi Jungyuen Franzin Carla M Marassi Francesca M |
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Institution: | The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. |
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Abstract: | An Escherichia coli plasmid vector for the high-level expression of hydrophobic membrane proteins is described. The plasmid, pBCL, directs the expression of a target polypeptide fused to the C terminus of a mutant form of the anti-apoptotic Bcl-2 family protein, Bcl-XL, where the hydrophobic C terminus has been deleted, and Met residues have been mutated to Leu to facilitate CNBr cleavage after a single Met inserted at the beginning of the target sequence. Fusion protein expression is in inclusion bodies, simplifying the protein purification steps. Here we report the high-level production of PLM, a membrane protein that is a member of the FXYD family of tissue-specific and physiological-state-specific auxiliary subunits of the Na,K-ATPase, expressed abundantly in heart and skeletal muscle. We demonstrate that milligram quantities of pure, isotopically labeled protein can be obtained easily and in little time with this system. |
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Keywords: | membrane protein expression FXYD PLM Mat-8 CHIF Bcl-XL NMR lipid |
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