Production and characterization of a retinoic acid receptor RAR纬 construction encompassing the DNA binding domain and the disordered N-terminal proline rich domain
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- 作者：Denise Martinez-Zapien ; Marc-Andr茅 Delsuc ; Gilles Trav茅 ; R茅gis Lutzing ; C茅cile Rochette-Egly ; Bruno Kieffer
- 关键词：Retinoic acid receptor ; Proline rich domain ; Intrinsically disordered proteins
- 刊名：Protein Expression and Purification
- 出版年：March, 2014
- 年：2014
- 卷：95
- 期：Complete
- 页码：113-120
- 全文大小：1622 K
文摘
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 15N, 13C 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 RAR纬 is disordered in solution, a state that is compatible with modifications such as phosphorylation.