Ni nta affinity column

The recombinant plasmids were individually transformed into E. coli BL21 Rosetta (DE3) or co-transformed together to express either individual proteins or the BMAL1/HIF2A complex. Expression of the proteins or BMAL1/HIF2A complex was carried out at 18 °C by induction with 0.1 mM IPTG (isopropyl-β-d-thiogalactoside) for 16 hours. The BMAL1/HIF2A heterodimer (including bHLH, PAS-A, and PAS-B domains) was first purified through a Ni-NTA affinity column (ThermoFisher Scientific, 88222) followed by Heparin chromatography. The heterodimer eluted from the Heparin column was further analyzed by size exclusion chromatography using a Superdex 200 Increase 10/300 GL column. The peak fractions were assessed using SDS-PAGE and Coomassie blue staining. Subsequently, they were combined and concentrated. The concentrated protein was then used for cryo-EM sample preparation or stored at −80 °C for further use. His-HIF2A and His-HIF1A were purified individually using Ni-NTA affinity columns, followed by dialysis to eliminate imidazole. The proteins were concentrated and stored at −80 °C for further use.

Escherichia coli BL21 (DE3) cells containing recombinant pET-28a (+) plasmids were cultured in Luria-Bertani (LB) medium with kanamycin (50 mg·L⁻¹) at 37 °C and with shaking at 200 rpm for 2 h. Enzyme expression was induced with IPTG (final concentration = 0.2 mM) when the bacteria reached OD₆₀₀ = 0.6–0.8. The cells were incubated at 20 °C and with shaking at 200 rpm for 12 h. His-tagged enzymes were purified with an ÄKTA purifier system (GE Healthcare, Little Chalfont, UK). The cells were resuspended in buffer A (100 mM potassium phosphate, 150 mM NaCl, and 20 mM imidazole; pH 7.5) and disrupted with an ultrasonic cell disruption system (SCIENTZ-IID; Ningbo Scientz Biotechnology, Ningbo, Zhejiang, China) followed by centrifugation at 12,000 rpm and 4 °C for 30 min to remove cell debris and obtain cell-free extracts. The latter were passed through a 0.22-μm filter and loaded into a standard Ni-NTA affinity column (Thermo Fisher Scientific, Waltham, MA, USA) pre-equilibrated with buffer A. The column was then gradient-eluted with buffer B (100 mM potassium phosphate, 150 mM NaCl, and 500 mM imidazole; pH 7.5). Eluted fraction purity was determined by SDS-PAGE.

Escherichia coli BL21 (DE3) cells containing recombinant pET-28a (+) plasmids were cultured in Luria–Bertani (LB) medium with kanamycin (50 mg L⁻¹) at 37 °C and with shaking at 200 rpm for 2 h. Enzyme expression was induced with IPTG (final concentration = 0.2 mM) when the bacteria reached OD₆₀₀ = 0.6–0.8. The cells were incubated at 20 °C and with shaking at 200 rpm for 12 h. His-tagged enzymes were purified with an ÄKTA purifier system (GE Healthcare, Little Chalfont, UK). The cells were resuspended in buffer A (100 mM potassium phosphate, 150 mM NaCl, and 20 mM imidazole; pH 7.5) and disrupted with an ultrasonic cell disruption system (SCIENTZ-IID; Ningbo Scientz Biotechnology, Ningbo, Zhejiang, China) followed by centrifugation at 12,000 rpm and 4 °C for 30 min to remove cell debris and obtain cell-free extracts. The latter were passed through a 0.22-μm filter and loaded into a standard Ni–NTA affinity column (Thermo Fisher Scientific, Waltham, MA, USA) pre-equilibrated with buffer A. The column was then gradient-eluted with buffer B (100 mM potassium phosphate, 150 mM NaCl, and 500 mM imidazole; pH 7.5). Eluted fraction purity was determined by SDS-PAGE (Additional file 2: Figure S9).