Bl21 de3 gold cells

Proteins were expressed in E. coli and purified as described previously (27 (link)). Briefly, n-terminally tagged 6xHis-TEV-PPARg LBD (PPARγ isoform 1/2 numbering: 202/230–477/505), 6xHis-PPARγ (isoform 2, 1-505), or 6xHis-TEV-RxRα was expressed in BL21-De3 Gold cells (Invitrogen) in either Luria-Bertani broth (PPARγ LBD) or terrific broth (PPARγ and RxRα full-length). Cells were grown at 37 °C to an OD of 0.8, dropped to 22 °C for 1 h, and then induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 16 h. Cells were harvested and lysed in 50 mM KPO4 (pH 8.0), 300 mM KCl, 1 mM TCEP, and 1 mM EDTA using a C-5 Emulsiflex high-pressure homogenizer (Avestin). Clarified lysate was flowed through two Histrap FF 5 mL columns in series (GE Healthcare). PPARγ LBD was incubated with 6xHis-tagged TEV overnight and passed through Histrap FF columns again to remove the 6xHis tag and TEV protease. Full-length protein was not cleaved with TEV. Size exclusion chromatography was then performed using a Hiload 16/600 Superdex 75 pg column (GE Healthcare), and purity was confirmed by SDS-page.