Bcl-XL as a fusion protein for the high-level expression of membrane-associated proteins.
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.
Thai
Khang
K
The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
Choi
Jungyuen
J
Franzin
Carla M
CM
Marassi
Francesca M
FM
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D051020:bcl-X Protein
D008565:Membrane Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051020:bcl-X Protein
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