Purification of Native and Recombinant NPC1

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Title: Purification of Native and Recombinant NPC1
Author: Dale, Jarrod Donald
Abstract: 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 .
URI: http : / /hdl .handle .net /2152 .5 /434
Date: 2008-12-23

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Purification of Native and Recombinant NPC1. Southwestern Medical School. Available electronically from http : / /hdl .handle .net /2152 .5 /434 .

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