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