Method for isolating an intestinal cholesterol binding protein
The invention describes a method for isolating an intestinal protein which is able to bind cholesterol and/or cholesterol uptake inhibitors.
1. A method for isolating a 145 kDa protein capable of specifically binding cholesterol absorption inhibitors, comprising the steps of:
i) incubating mammalian intestinal tissue or cells with a photoreactive inhibitor of cholesterol uptake which is radioactively labeled and biotin-tagged,
ii) solubilizing said intestinal cells and separating the supernatant from the cell debris,
iii) adding said supernatant onto streptavidin-agarose,
iv) adding the eluate of said streptavidin-agarose onto a SDS-PAGE and
v) excising the proteins running in a size range of 140 to 150 kDa and eluting said proteins,
vi) isolating a complex comprising the 145 kDa protein bound to the photoreactive inhibitor of cholesterol uptake and
vii) separating the 145 kDa protein from the cholesterol absorption inhibitor,
wherein the photoreactive inhibitor of cholesterol uptake which is biotin tagged has the following structure:
2. The method of claim 1 wherein said intestinal tissue or cells are from an intestinal cell culture or ileal brush border cells from human, rat, mouse or rabbit.
3. A method for isolating a complex formed by a purified 145 kDa protein capable of specifically binding cholesterol absorption inhibitors and a cholesterol absorption inhibitor, comprising the steps of:
i) incubating mammalian intestinal tissue or cells with a cholesterol absorption inhibitor that is a photoreactive inhibitor of cholesterol uptake which is radioactively labeled and biotin-tagged,
ii) solubilizing said intestinal cells and separating the supernatant from the cell debris,
iii) adding said supernatant onto streptavidin-agarose,
iv) adding the eluate of said streptavidin-agarose onto a SDS-PAGE and
v) excising the proteins running in a size range of 140 to 150 kDa and eluting said proteins bound to said cholesterol absorption inhibitor , and
iv) isolating the complex,
said cholesterol absorption inhibitor having the following structure
4. A method for isolating a complex formed by a purified 145 kDa protein capable of specifically binding cholesterol absorption inhibitors and a cholesterol absorption inhibitor, comprising the steps of:
i) incubating mammalian intestinal tissue or cells with a photoreactive inhibitor of cholesterol uptake which is biotin-tagged,
ii) solubilizing said intestinal cells and separating the supernatant from the cell debris,
iii) adding said supernatant onto streptavidin-agarose,
iv) adding the eluate of said streptavidin-agarose onto a SDS-PAGE and
v) excising the proteins running in a size range of 140 to 150 kDa and eluting said proteins bound to said cholesterol absorption inhibitor, and
iv) isolating the complex,
said photoreactive inhibitor having the following structure of Formula I:
wherein R shall be selected from one of the following groups a) and b):