Patent
Method for isolating an intestinal cholesterol binding protein
| العنوان: | Method for isolating an intestinal cholesterol binding protein |
|---|---|
| Patent Number: | 7,786,103 |
| تاريخ النشر: | August 31, 2010 |
| Appl. No: | 11/872855 |
| Application Filed: | October 16, 2007 |
| مستخلص: | The invention describes a method for isolating an intestinal protein which is able to bind cholesterol and/or cholesterol uptake inhibitors. |
| Inventors: | Kramer, Werner (Mainz-Laubenheim, DE); Frick, Wendelin (Hunstetten-Beuerbach, DE) |
| Assignees: | Sanofi-Aventis Deutschland GmbH (Frankurt am Main, DE) |
| Claim: | 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: [chemical expression included] |
| Claim: | 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. |
| Claim: | 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 [chemical expression included] |
| Claim: | 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: [chemical expression included] wherein R shall be selected from one of the following groups a) and b): [chemical expression included] |
| Current U.S. Class: | 51421/002 |
| Patent References Cited: | 5264372 November 1993 Beaumont et al. 5698445 December 1997 Abo et al. 6225454 May 2001 Miyagi et al. 6383734 May 2002 Marshall et al. 2002/0009714 January 2002 Altmann et al. 2002/0039774 April 2002 Kramer et al. 0245979 November 1987 2000166545 June 2000 90/05147 May 1990 91/18098 November 1991 00/60062 October 2000 WO 00/63703 October 2000 WO 02/18432 March 2002 |
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| Assistant Examiner: | Kosson, Rosanne |
| Primary Examiner: | Carlson, Karen Cochrane |
| رقم الانضمام: | edspgr.07786103 |
| قاعدة البيانات: | USPTO Patent Grants |
| الوصف غير متاح. |