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Related Experiment Videos

Pure and functionally homogeneous recombinant retinoid X receptor

Z P Chen1, L Shemshedini, B Durand

  • 1Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS et Unité 184 de Biologie Moléculaire et de Génie Génétique de de l'INSERM, Institut de Chimie Biologique, Faculté de Médecine, Strasbourg, France.

The Journal of Biological Chemistry
|October 14, 1994
PubMed
Summary

Researchers purified mouse retinoid X receptor alpha (RXR alpha) lacking regions A/B, finding it binds 9-cis-retinoic acid (9-cis-RA) but shows reduced DNA binding compared to animal cell-expressed versions.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Receptor Biology

Background:

  • Retinoid X receptor alpha (RXR alpha) is a nuclear receptor crucial for gene regulation.
  • The amino-terminal region A/B of RXR alpha plays a role in its function.
  • Understanding RXR alpha's ligand binding and DNA interaction is key to deciphering its biological roles.

Purpose of the Study:

  • To purify and characterize a truncated form of mouse RXR alpha (RXR alpha delta AB) lacking the amino-terminal A/B region.
  • To investigate the ligand-binding properties of purified RXR alpha delta AB with 9-cis-retinoic acid (9-cis-RA).
  • To compare the DNA-binding efficiency of recombinant RXR alpha delta AB expressed in different systems (bacterial, baculovirus, animal cells).

Main Methods:

  • Recombinant expression of RXR alpha delta AB in bacterial and baculovirus systems.

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  • Protein purification to >98% purity and functional homogeneity.
  • Fluorescence quenching analysis for ligand-binding affinity and stoichiometry.
  • DNA-binding assays using direct repeat 1 (DR1) elements.

    • Main Results:

    • Purified RXR alpha delta AB from bacterial and baculovirus systems was soluble and bound 9-cis-RA with high affinity.
    • Bacterial and baculovirus-expressed RXR alpha delta AB showed significantly lower DNA binding efficiency (10-fold and 5-fold, respectively) compared to COS-1 cell-expressed protein.
    • 9-cis-RA did not stimulate DR1 binding for RXR alpha delta AB expressed in E. coli, Sf9, or COS-1 cells.
    • The observed ligand effect in previous studies might stem from conformational stabilization of improperly folded in vitro synthesized protein.

    Conclusions:

    • Recombinant RXR alpha delta AB can be functionally expressed and purified from prokaryotic and eukaryotic systems.
    • Animal cell-specific factors or modifications likely enhance RXR alpha's DNA-binding efficiency.
    • The ligand-dependent modulation of DNA binding observed in vitro may be an artifact of protein folding rather than a true biological mechanism.