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Purification and Characterization of Human Niemann–Pick C1 Protein

  • Xin GongEmail author
  • Hongwu Qian
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1949)

Abstract

Niemann–Pick C1 (NPC1) is a membrane protein required for the transport of low-density lipoprotein (LDL)-derived cholesterol from endosomes and lysosomes to the other organelles. Here, we describe the recombinant protein expression, purification, and characterization of the human NPC1. The protein is transiently expressed in human embryonic kidney (HEK) cells. Our purification protocol describes the steps to obtain a pure and homogeneous NPC1 protein. Niemann–Pick C2 (NPC2) is a small soluble protein, which mediates cholesterol transport in tandem with NPC1. Finally, we also describe two biochemical approaches to characterize NPC1 function in vitro—a cholesterol transfer assay from purified NPC2 to NPC1 and a binding assay between NPC1 and NPC2.

Key words

Niemann–Pick disease type C NPC1 NPC2 Cholesterol transport Membrane protein Transient expression Protein purification Cholesterol transfer 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Molecular BiologyPrinceton UniversityPrincetonUSA
  2. 2.Department of BiologySouthern University of Science and TechnologyShenzhenChina

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