1. Growth and Purification of hFXR LBD Abby McArthur Dr. 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 farnesoid X 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 = )
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 # 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