Growth and Purification
of hFXR LBD
Abby McArthur
Dr. Victor Hsu
Background Information
• Metabolic disordersdiabetes, 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.
Growth process
E. coli BL 21(DE3) pLysS
pET
PLysS
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
mgml
15N His-tagged hFXRLBD
Talon Co++
His-tagged Purification Resin
6 Liters Cultured
Buffer
50 mM NaP pH 7.3
0.5 M NaCl
0.5 M Sucrose
0.5 mM CHAPS
6/30/10
1.4
1.2
1.0
mg/ml
0.8
0.6
0.4
0.2
0.0
3
4
5
6
7
8
Fractions
9
10
11
12
• 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
Induce immediately
#1
Wait 1 hr
#2
Set pellet on bench 30 min
Take up pellet (M9),
induce immediately
#3
Take up pellet, wait 1 hr
and induce
#4
Results: 7/14/10 expt.
Results: 7/28/10 expt.
2 liter culture
Induced at 20 degrees Celsius for
8 hours
Total protein yield : 7.96 mg
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