Bacterial expression, refolding, functional characterization, and mass spectrometric identification of full-length human PPAR-gamma.
A systematic strategy for producing biologically active full-length human peroxisome proliferator-activated receptor-gamma (PPAR-gamma) was developed. PPAR-gamma was expressed as inclusion bodies in Terrific Broth (TB) ensuring stable pH, better growth conditions, and 4-fold higher cell yield as compared to Luria Broth (LB). Purification was performed by a combination of immobilized metal-ion affinity chromatography (IMAC) and size exclusion chromatography (SEC), yielding 176 mg of PPAR-gamma of over 90% purity per liter of TB. A simplified refolding setup, capable of gradual buffer exchange and continuous protein feeding, was used to refold the denatured PPAR-gamma with approximately 66% yield. Correct refolding of the denatured PPAR-gamma was assessed with non-denaturing gels and SEC. The refolded PPAR-gamma displayed its ligand binding ability for rosiglitazone at K(d)=250+/-6 nM as determinated by SEC-HPLC assay. In addition, DNA binding activity of the refolded PPAR-gamma was demonstrated by electrophoretic mobility shift assay (EMSA) using a PPRE motif. The integrity of PPAR-gamma was confirmed by mass spectrometry. Our results indicate the feasibility of using these strategies to produce biologically active full-length PPAR-gamma in E. coli BL21 (DE3).
Department of Medicinal Chemistry, School of Pharmacy, Chongqing Medical University, Chongqing, China.
Wei
W
Li
Yonghua
Y
Yuan
Zongwei
Z
Luo
Xiaohong
X
Zheng
Ling
L
Zhao
Wen
W
Duan
Yu
Y
Yu
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D047495:PPAR gamma / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020673:Protein Renaturation
D047495:PPAR gamma