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 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

Xanthohumol

• Prenylflavonoid

• Found in hops

• Thought to express SBARM-like activity

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.

pET

PLysS

E. coli BL 21(DE3) pLysS

Growth process

pET 15B vector containing hFXR gene

Induction

Purification• Buffer solution with protease inhibitors

• Cells sonicated, centrifuged, and supernatant saved

• Mixed with cobalt polyhistidine affinity resin

Histidine

Column chromatography

• Solution run through a column

• Eluted with imidazole (first 10 mM, then 200 mM)

• Imidazole displaces the his-tag proteins

Imidazole Histidine

Protein concentration 6/30/10

3 4 5 6 7 8 9 10 11 12

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

mg

/ml

Fractions

mgml15N His-tagged hFXRLBD Talon Co++ His-tagged Purification Resin6 Liters CulturedBuffer 50 mM NaP pH 7.30.5 M NaCl0.5 M Sucrose0.5 mM CHAPS6/30/10

• Total protein yield: 5 mg

Gel Results: 6/30/10

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

Results: 7/14/10 expt.

Results: 7/28/10 expt.

Total protein yield : 7.96 mg

2 liter culture

Induced at 20 degrees Celsius for 8 hours

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

Aknowledgements• Howard Hughes Medical Institute (HHMI)

• Dr. Victor Hsu, mentor

• Dr. Dave Broderick

• Dr. Claudia Maier

• Dr. Kevin Ahern, advisor