CONSTRUCTIONAL ENGINEERING ASSOCIATION – LABOUR BROKING DIVISION (CEA - LBD)
Growth and Purification of hFXR LBD Abby McArthur Dr. Victor Hsu
description
Transcript of Growth and Purification of hFXR LBD Abby McArthur Dr. Victor Hsu
Growth and Purification of hFXR LBD
Abby McArthurDr. 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