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Growth and Purification of hFXR LBD Abby McArthur Dr. Victor Hsu

Growth and Purification of hFXR LBD Abby McArthur Dr. Victor Hsu. Background Information. Metabolic disorders-diabetes, stroke and heart disease-affect up to 25% of the U.S. population. Transcription factors. Human farnesoid X r eceptor ( hFXR ). Binds to DNA

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Growth and Purification of hFXR LBD Abby McArthur Dr. Victor Hsu

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  1. Growth and Purification of hFXR LBDAbby McArthurDr. Victor Hsu

  2. Background Information • Metabolic disorders-diabetes, stroke and heart disease-affect up to 25% of the U.S. population • Transcription factors

  3. Human farnesoidX receptor (hFXR) • Binds to DNA • Regulates synthesis of triglycerides, lipoprotein metabolism, and glucose metabolism • Ligand-binding domain (LBD) • FXR-targeting ligands still being researched (SBARMs) • Agonist/antagonist

  4. Xanthohumol • Prenylflavonoid • Found in hops • Thought to express SBARM-like activity

  5. Hypothesis • Our hypothesis is that XN does in fact bind directly to FXR and that it promotes distinct structural and conformational changes in FXR, resulting in SBARM-like activity.

  6. Growth process E. coli BL 21(DE3) pLysS pET 15B vector containing hFXR gene pET PLysS

  7. Induction

  8. Purification • Buffer solution with protease inhibitors • Cells sonicated, centrifuged, and supernatant saved • Mixed with cobalt polyhistidine affinity resin Histidine

  9. Column chromatography • Solution run through a column • Eluted with imidazole (first 10 mM, then 200 mM) • Imidazole displaces the his-tag proteins Imidazole Histidine

  10. Protein concentration 6/30/10 • Total protein yield: 5 mg

  11. Gel Results: 6/30/10

  12. How to get more protein? • An experiment with four samples was designed as follows: • 1 L culture • Inoculation (put in shaker) Centrifuge (when A600 = 0.6-0.7) Take up pellet + M9 Set pellet on bench 30 min Induce immediately Wait 1 hr Take up pellet (M9),Take up pellet, wait 1 hr #1 #2 induce immediately and induce #3 #4

  13. Results: 7/14/10 expt.

  14. Results: 7/28/10 expt. 2 liter culture Induced at 20 degrees Celsius for 8 hours Total protein yield : 7.96 mg

  15. Future applications • Grow and purify more protein • Create small molecule database of other known FXR ligands • Bind known ligands as well as XN to FXR and analyze through NMR spectroscopy

  16. Aknowledgements • Howard Hughes Medical Institute (HHMI) • Dr. Victor Hsu, mentor • Dr. Dave Broderick • Dr. Claudia Maier • Dr. Kevin Ahern, advisor

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