Expression and Purification of

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Expression and Purification of mCAR hRXR DNA
Binding Domain Spring 2005 Lab Rotation
Presentation By Sangeetha Rajagopalan (GST)
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Nuclear Receptors

• Family of structurally similar, ligand-activated
  transcription factors
• Essential for normal physiology, differentiation,
  development, homeostasis
• Transcriptional activity- small lipophilic
  ligands
• Common modular structure
• DNA binding domain(DBD)
• Ligand binding domain(LBD)
• DBD recognize specific, regulatory DNA sequence
• LBD highly conserved C terminal, conformational
  change on binding of agonist ligand

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Tissue Distribution of Common Nuclear Receptors
Liver
Intestine
Kidney
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Constitutive
Androstane
Receptor
Transcriptional activation of genes

v
xenobiotic
steroid
-
metabolizing enzyme
v
Expressed

liver, intestinal epithelium,
stomach
v
Highly conserved DBD
v
Hinge domain
v
Divergent ligand
-
binding/dimerization/transcriptional activation
domain
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Retinoid X Receptor
v
Ligand
-
dependent transcription factor
v
Endogenous
ligand

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cis
retinoic
acid
v
Role

fundamental biological
process reproduction, cellular
differentiation,
hematopoesis
v
Regulate activity of other nuclear
hormone receptors, form
heterodimers
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Mechanism of CAR-RXR

• CAR-RXR heterodimer
• CAR interacts with DNA as a heterodimer with RXR
• Form heterodimer with RXR bind to Response
  elements(REs)-DR4-type
• The heterodimeric complex regulate genes

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Activation of Nuclear Receptors
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Protein Purification

• Pellet resuspended in lysis buffer
• Cells lysed using French press 1500 Pa
• Lysed centrifuged, supernatant PH 7.4
• DTT added to supernatant
• Loaded onto glutathione-sepharose 4B column
• Protein eluted using 10mM reduced glutathione( in
  1X thrombin cleavage buffer)
• Eluted protein dialyzed
• GST tag cleaved with thrombin protease
• Cleaved protein subjected to gel filtration
• Fractions obtained according molecular weight
• Analyzed on SDS-PAGE
• GST-hRXR DBD - 36,970Da GST-mCAR DBD
  37,485Da
• GST - 26,000Da
• hRXR DBD - 10,970.5242Da mCAR DBD
  11,485.4978Da

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Gel-filtration chromatogram SDS-PAGE of hRXR DBD
FractionNo.
Absorbance (absobance unit)
Elution time
hRXR DBD
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Gel-filtration chromatogram SDS-PAGE of mCAR DBD
Fraction No.
Absorbance (absorbance units)
Elution time
mCAR DBD
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Acknowledgement

• Dr. Elias Fernandez
• Jeremy Vincent
• Dr. Li Shan