Production and characterization of a retinoic acid receptor RARγ construction encompassing the DNA binding domain and the disordered N-terminal proline rich domain

Protein Expr Purif. 2014 Mar;95:113-20. doi: 10.1016/j.pep.2013.12.001. Epub 2013 Dec 13.

Abstract

Gene activation by retinoic acid nuclear receptors (RAR) is regulated by a number of molecular events such as ligand binding, interaction with cognate DNA sequences and co-regulatory proteins, and phosphorylation. Among the several phosphorylation sites that are involved in the non-genomic regulatory pathways of the RAR, two are located in a proline rich domain (PRD) within the N-terminal domain (NTD) of the receptor. This region is predicted to be intrinsically disordered, complicating its production and purification. We present here an approach enabling the high yield production of RAR fragments encompassing the PRD and the DNA binding domain (DBD). We found that expression levels were dependent on where the position of the N-terminal boundary of the fragment was placed within the RAR sequence. The purification protocol involves the use of maltose binding protein as a solubilising tag and extensive centrifugation steps at critical points of the purification process. This protocol is suitable to express (15)N, (13)C labeled proteins enabling nuclear magnetic resonance studies. The resulting proteins were characterized by biophysical methods including Small Angle X-ray Scattering and NMR. These studies showed that PRD extension of RARγ is disordered in solution, a state that is compatible with modifications such as phosphorylation.

Keywords: Intrinsically disordered proteins; Proline rich domain; Retinoic acid receptor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • DNA / chemistry
  • DNA / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Humans
  • Intrinsically Disordered Proteins / chemistry
  • Intrinsically Disordered Proteins / genetics
  • Intrinsically Disordered Proteins / metabolism
  • Mice
  • Molecular Sequence Data
  • Proline
  • Protein Structure, Tertiary
  • Receptors, Retinoic Acid / chemistry*
  • Receptors, Retinoic Acid / genetics
  • Receptors, Retinoic Acid / metabolism*
  • Recombinant Proteins / chemistry*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism*
  • Sequence Alignment

Substances

  • Intrinsically Disordered Proteins
  • Receptors, Retinoic Acid
  • Recombinant Proteins
  • retinoic acid receptor gamma
  • DNA
  • Proline