Purification of Native and Recombinant NPC1




Dale, Jarrod Donald

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The Niemann-Pick, Type C1 protein (NPC1) is required for the transport of lipoproteinderived cholesterol from lysosomes to endoplasmic reticulum. The 1278-amino acid, polytopic membrane protein has not been purified, and its mechanism of action is unknown. We encountered NPC1 in a search for a membrane protein that binds 25-hydroxycholesterol (25-HC) and other oxysterols. Described here is the initial purification of rabbit NPC1 using a classical biochemical approach and an analysis of the sterol binding properties of native and recombinant NPC1. Our purification yielded a membrane-bound 25-HC-binding protein which was purified more than 14,000-fold from rabbit liver membranes. This protein was identified as NPC1 by mass spectroscopy. We prepared recombinant human NPC1 and confirmed its ability to bind oxysterols, including those with a hydroxyl group on the 24, 25, or 27 positions. Hydroxyl groups on the 7, 19, or 20 positions failed to confer binding. Initial characterization of the sterol binding properties showed specific binding for 25-HC; however, we were unable to demonstrate significant binding of NPC1 to cholesterol using our current experimental conditions. The availability of assays to measure NPC1 sterol binding in vitro may further the understanding of intracellular sterol transport.

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