Purification of HIF-2α-ARNT Complex

To obtain HIF-2α-ARNT complex proteins, the pSJ2-HIF-2α plasmid was co-transformed along with pMKH-ARNT into BL21-CodonPlus (DE3)-RIL competent cells (Agilent Technologies). Following 0.1 mM IPTG induced protein expression overnight at 16 °C, cell pellets were lysed by sonication, and supernatants were applied onto pre-packed His∙Bind resin (Novagen). The bound proteins were further purified using SP Sepharose (GE Healthcare), and the eluted fractions were then loaded on a HiLoad 16/60 Superdex 200pg gel filtration column (GE Healthcare) equilibrated in 20 mM Tris (pH 8.0) and 400 mM NaCl. DTT was added to the pooled protein peak fractions at 10 mM. The heterodimeric proteins of HIF-2α and ARNT-GFP were prepared similarly as described above, except that the pMKH-ARNT-GFP plasmid was used in the place of pMKH-ARNT. The ARNT-GFP protein was co-expressed and purified in complex with HIF-1α and NPAS3 (plasmids made in our previous studies^{3 (link),8 (link)}), respectively. The single PAS-B domain of HIF-2α was produced by transformation of pSJ2-HIF-2α (241–361) into BL21-CodonPlus (DE3)-RIL, followed by overnight expression, and purification using His-tag affinity chromatography and gel filtration chromatography.