Bacterial expression, refolding, functional characterization, and mass spectrometric identification of full-length human PPAR-γ

Abstract

A systematic strategy for producing biologically-active full-length human peroxisome proliferator-activated receptor-γ (PPAR-γ) was developed. PPAR-γ 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-γ 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-γ with approximately 66% yield. Correct refolding of the denatured PPAR-γ was assessed with non-denaturing gels and SEC. The refolded PPAR-γ displayed its ligand binding ability for rosiglitazone at Kd = 250 ± 6nM as determinated by SEC-HPLC assay. In addition, DNA binding activity of the refolded PPAR-γ was demonstrated by electro-phoretic mobility shift assay (EMSA) using a PPRE motif. The integrity of PPAR-γ was confirmed by mass spectrometry. Our results indicate the feasibility of using these strategies to produce biologically active full-length PPAR-γ in E. coli BL21 (DE3).