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Protein Sci. 2015 Dec;24(12):2008-19. doi: 10.1002/pro.2812. Epub 2015 Oct 14.

Expression, purification, and functional characterization of the insulin-responsive facilitative glucose transporter GLUT4.

Author information

1
Department of Pediatrics, Washington University School of Medicine, St Louis, Missouri, 63110.
2
Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, Missouri, 63110.

Abstract

The insulin-responsive facilitative glucose transporter GLUT4 is of fundamental importance for maintenance of glucose homeostasis. Despite intensive effort, the ability to express and purify sufficient quantities of structurally and functionally intact protein for biophysical analysis has previously been exceedingly difficult. We report here the development of novel methods to express, purify, and functionally reconstitute GLUT4 into detergent micelles and proteoliposomes. Rat GLUT4 containing FLAG and His tags at the amino and carboxy termini, respectively, was engineered and stably transfected into HEK-293 cells. Overexpression in suspension culture yielded over 1.5 mg of protein per liter of culture. Systematic screening of detergent solubilized GLUT4-GFP fusion protein via fluorescent-detection size exclusion chromatography identified lauryl maltose neopentyl glycol (LMNG) as highly effective for isolating monomeric GLUT4 micelles. Preservation of structural integrity and ligand binding was demonstrated via quenching of tryptophan fluorescence and competition of ATB-BMPA photolabeling by cytochalasin B. GLUT4 was reconstituted into lipid nanodiscs and proper folding was confirmed. Reconstitution of purified GLUT4 with amphipol A8-35 stabilized the transporter at elevated temperatures for extended periods of time. Functional activity of purified GLUT4 was confirmed by reconstitution of LMNG-purified GLUT4 into proteoliposomes and measurement of saturable uptake of D-glucose over L-glucose. Taken together, these data validate the development of an efficient means to generate milligram quantities of stable and functionally intact GLUT4 that is suitable for a wide array of biochemical and biophysical analyses.

KEYWORDS:

FSEC; GLUT4; detergent screening; glucose transport; liposomes; mammalian membrane protein; nanodiscs; protein expression

PMID:
26402434
PMCID:
PMC4815238
DOI:
10.1002/pro.2812
[Indexed for MEDLINE]
Free PMC Article

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