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Bcl-XL as a fusion protein for the high-level expression of membrane-associated proteins

The Burnham Institute, La Jolla, California 92037, USA

(RECEIVED November 19, 2004; FINAL REVISION December 22, 2004; ACCEPTED December 22, 2004)

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.

Keywords: membrane protein expression; FXYD; PLM; Mat-8; CHIF; Bcl-XL; NMR; lipid

Abbreviations: BCL, Bcl-XL mutant lacking the hydrophobic C terminus adapted as fusion partner • Bcl-2, B-cell leukemia/lymphoma 2 • Bcl-XL, B-cell leukemia/lymphoma extra long • CHIF, corticosteroid-hormone-induced factor, FXYD4 • CNBr, cyanogen bromide • CPMOIST, cross polarization with mismatch-optimized IS polarization transfer • DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine • DOPG, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] • GdnHCl, guanidinium HCl • HPLC, high-performance liquid chromatography • HSQC, heteronuclear single quantum coherence • HSQC-NOESY, HSQC–nuclear overhauser effect spectroscopy • IPTG, isopropyl -D-thiogalactopyranoside • KSI, ketosteroid isomerase • ER, endoplasmic reticulum • MALDI-TOF, matrix-assisted laser desorption ionization–time of flight • Mat-8, mammary tumor protein 8 kDa, FXYD3 • NMR, nuclear magnetic resonance • PISEMA, polarization inversion with spin exchange at the magic angle • PLM, phospholemman, FXYD1 • PAGE, polyacrylamide gel electrophoresis • pI, isoelectric point • SDS, sodium dodecyl sulfate • TLE, a portion of the Trp LE 1413 polypeptide

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041244305.

Reprint requests to: Francesca M. Marassi, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA; e-mail: fmarassi{at}burnham.org; fax: (858) 713-6281.

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