Method for isolating an intestinal cholesterol binding protein
AssigneeSanofi-Aventis Deutschland GmbH
InventorsWerner KRAMER, Wendelin FRICK
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Abstract
The invention describes a method for isolating an intestinal protein which is able to bind cholesterol and/or cholesterol uptake inhibitors.
Claims5
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 are 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
- wherein the photoreactive inhibitor of cholesterol uptake which is biotin tagged has the following structure:
A method for isolating a complex formed by a purified 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.
- and said compound having the following structure
A method for isolating a complex formed by a purified 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 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 and said photoreactive inhibitor having the following structure of Formula I
- wherein R shall be selected from one of the following groups a) and b):
A method for isolating a complex formed by a purified 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,
- ii) solubilizing said intestinal cells are and separating the supernatant from the cell debris,
- iii) subjecting said supernatant to chromatography,
- iv) adding the eluate onto SDS-PAGE and,
- v) excising the proteins running in a size range of 140 to 150 kDa and eluting said proteins and said photoreactive inhibitor having the following structure of Formula I
- wherein R shall be selected from one of the following groups a) and b):
Dependencies are inferred from the claim text.