PMID- 23562662
OWN - NLM
STAT- MEDLINE
DCOM- 20131127
LR  - 20161125
IS  - 1096-0279 (Electronic)
IS  - 1046-5928 (Linking)
VI  - 89
IP  - 2
DP  - 2013 Jun
TI  - Transient expression, purification and characterisation of human full-length 
      PPARgamma2 in HEK293 cells.
PG  - 189-95
LID - S1046-5928(13)00057-0 [pii]
LID - 10.1016/j.pep.2013.03.012 [doi]
AB  - 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 (PPARgamma). As a multidomain 
      transcription factor, PPARgamma 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 PPARgamma isoforms (PPARgamma1 and PPARgamma2) as pure full-length 
      proteins in adequate quantity has hindered detailed mechanistic studies of PPARgamma 
      and its ancillary protein partners. Here we report an efficient transient 
      expression system to produce recombinant human full-length PPARgamma2 protein. The 
      DNA encoding the human full-length PPARgamma2 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 
      PPARgamma2 protein was confirmed by mass spectrometry analysis. The purified PPARgamma2 
      protein was active in ligand binding and could be phosphorylated in vitro by 
      Cdk5/p25 at Ser 273. Further studies showed that selected PPARgamma modulators 
      inhibited Cdk5-mediated PPARgamma2 Ser 273 phosphorylation in vitro. Our results 
      demonstrate the feasibility of producing large quantities of pure and functional 
      human full-length PPARgamma2 suitable for drug discovery applications.
CI  - Copyright (c) 2013 Elsevier Inc. All rights reserved.
FAU - Liu, Jianming
AU  - Liu J
AD  - Discovery Sciences, AstraZeneca R&D, Pepparedsleden 1, 43183 Molndal, Sweden. 
      Jianming.Liu@Astrazeneca.com
FAU - Ormo, Mats
AU  - Ormo M
FAU - Nystrom, Ann-Christin
AU  - Nystrom AC
FAU - Claesson, Josefine
AU  - Claesson J
FAU - Giordanetto, Fabrizio
AU  - Giordanetto F
LA  - eng
PT  - Journal Article
DEP - 20130403
PL  - United States
TA  - Protein Expr Purif
JT  - Protein expression and purification
JID - 9101496
RN  - 0 (Ligands)
RN  - 0 (PPAR gamma)
RN  - 0 (Recombinant Proteins)
RN  - EC 2.7.11.1 (Cyclin-Dependent Kinase 5)
SB  - IM
MH  - Amino Acid Sequence
MH  - Cyclin-Dependent Kinase 5/metabolism
MH  - *Gene Expression
MH  - Genetic Vectors/genetics
MH  - HEK293 Cells
MH  - Humans
MH  - Ligands
MH  - Molecular Sequence Data
MH  - PPAR gamma/*chemistry/*genetics/isolation & purification/metabolism
MH  - Phosphorylation
MH  - Protein Binding
MH  - Recombinant Proteins/chemistry/genetics/isolation & purification/metabolism
EDAT- 2013/04/09 06:00
MHDA- 2013/12/16 06:00
CRDT- 2013/04/09 06:00
PHST- 2012/12/12 00:00 [received]
PHST- 2013/03/19 00:00 [revised]
PHST- 2013/03/25 00:00 [accepted]
PHST- 2013/04/09 06:00 [entrez]
PHST- 2013/04/09 06:00 [pubmed]
PHST- 2013/12/16 06:00 [medline]
AID - S1046-5928(13)00057-0 [pii]
AID - 10.1016/j.pep.2013.03.012 [doi]
PST - ppublish
SO  - Protein Expr Purif. 2013 Jun;89(2):189-95. doi: 10.1016/j.pep.2013.03.012. Epub 
      2013 Apr 3.