Purification of Niemann-Pick C2 protein (bNPC2) from bovine milk and investigation of cholesterol transfer role

In the present study, bovine Niemann-Pick C2 protein (bNPC2) is isolated from bovine milk by sequentially using salt precipitation, dialysis, DEAE-Sephadex A-50 anion exchange chromatography, CM-Sepharose and SP-Sepharose cation exchange chromatographies. bNPC2 is defined by using SDS-PAGE and Western-Blotting techniques. Donor vesicles composed of bis(monomyristoylglycerol) phosphate (20 % mol), 4-[14C]-cholesterol (1 % mol), cholesterol (10 % mol), N-[6-(biotinoyl)amino]hexanoyl-1,2-dihexa-decanoyl-sn-glycerol-3-phosphoethanolamine triethylammonium salt (4 % mol), phosphatidylcholine (65 % mol), and acceptor vesicles composed of bis(monomyristoylglycerol)phosphate (20 % mol), cholesterol (10 % mol), phosphatidylcholine (66 % mol), N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (4 % mol) were used in experiments to investigate the role and efficiency of bNPC2 in cholesterol transfer. It was determined that bNPC2 has an effective role in 4-[14C]-cholesterol transfer from donor to acceptor vesicles with a transfer efficiency of 52.3±5.2 %.