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Protein Expr Purif. 2013 Jun;89(2):189-95. doi: 10.1016/j.pep.2013.03.012. Epub 2013 Apr 3.

Transient expression, purification and characterisation of human full-length PPARγ2 in HEK293 cells.

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  • 1Discovery Sciences, AstraZeneca R&D, Pepparedsleden 1, 43183 Mölndal, Sweden. Jianming.Liu@Astrazeneca.com

Abstract

Effective anti-diabetic drugs known as thiazolidinediones (e.g. rosiglitazone, pioglitazone) exert their therapeutic effects through their agonistic activity at the peroxisome proliferator-activated receptor gamma (PPARγ). As a multidomain transcription factor, PPARγ forms heterodimers with different retinoid X receptors (RXRs) to modulate target gene expression at the transcriptional level in response to natural or synthetic ligands. Difficulties in producing either of the two major human PPARγ isoforms (PPARγ1 and PPARγ2) as pure full-length proteins in adequate quantity has hindered detailed mechanistic studies of PPARγ and its ancillary protein partners. Here we report an efficient transient expression system to produce recombinant human full-length PPARγ2 protein. The DNA encoding the human full-length PPARγ2 was cloned into a mammalian episomal vector and transiently expressed in human embryonic kidney 293 (HEK293-6E) cells with an expression level of 10mg/L culture. Identity of the purified recombinant PPARγ2 protein was confirmed by mass spectrometry analysis. The purified PPARγ2 protein was active in ligand binding and could be phosphorylated in vitro by Cdk5/p25 at Ser 273. Further studies showed that selected PPARγ modulators inhibited Cdk5-mediated PPARγ2 Ser 273 phosphorylation in vitro. Our results demonstrate the feasibility of producing large quantities of pure and functional human full-length PPARγ2 suitable for drug discovery applications.

Copyright © 2013 Elsevier Inc. All rights reserved.

PMID:
23562662
[PubMed - indexed for MEDLINE]
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