Gstrap ff

GST fusion protein containing pGEX-Klf4 or pGEX vector was induced with 0.1 mM of IPTG (Sigma-Aldrich). GST or GST-Klf4 was purified by GSTrap™ FF (GE Healthcare). Total cell lysates were incubated with GST beads (Biosciences, Sweden) overnight at 4 °C followed by four times washing using washing buffer. Finally, proteins were visible by western blot analysis.

The pGEX-2T/ERRα wt, Δ300aa, Δ398aa or pGEX-2T vector (GE Healthcare) was transformed into the BL21 strain (RBC Bioscience) and GST fusion protein expression was induced with 0.1 mM of IPTG (Nacalai Tesque). GST or GST-ERRα was purified using GSTrap™ FF (GE Healthcare). Whole cell lysates were incubated with GST-Accept (Nacalai Tesque) overnight at 4°C. Complexes were washed three times with washing buffer (50 mM Tris-HCl, pH 7.5) (Nacalai Tesque), 150 mM NaCl (Nacalai Tesque), 0.05% Tween20 (Wako)). Proteins interacting with ERRα were eluted with the sample buffer (2% SDS, 3% glycerol, 125 mM Tris-HCl, pH 6.5), 4% 2-mercaptoethanol, and 0.0125% BPB (all from Nacalai Tesque) and detected by western blot analysis.

ANXA1 cDNA was cloned into a pGEX-6P-1 expression vector. The protein was expressed as GST-ANXA1 fusion protein in E. coli BL21(DE3) cells obtained from New England Biolabs (Ipswich, England). Purification of the GST-ANXA1 from soluble E. coli extract was achieved by affinity chromatography using GSTrap FF (GE Health Care, Freiburg, Germany) according to the manufacturer’s instructions. After buffer exchange into PreScission protease cleavage buffer using a HiTrap desalting column (GE Healthcare, Freiburg, Germany), the GST tag was cleaved off upon incubation with PreScission protease (GE Healthcare). Subsequently, GST was removed by affinity capture using Glutathione Sepharose 4B (GE Healthcare, Freiburg, Germany). Residual amounts of GST were removed by incubation with GST Trap resin (ChromoTek, Martinsried, Germany). Finally, the buffer comprising cleaved ANXA1 was exchanged to 20 mM HEPES pH 6.0, 150 mM NaCl by dialysis using D-Tube Dialyzer (Merck Millipore, Massachusetts, US). Purified ANXA1 was stored at a concentration of 1.6 mg/mL at −78 °C. Purity and integrity of produced ANXA1 were examined by SDS PAGE followed by Coomassie staining and mass spectrometry.

We used a thermostable and cysteine-modified mutant of human p53 (C124A, C135V, C141V, W146Y, C182S, V203A, R209P, C229Y, H233Y, Y234F, N235K, Y236F, T253V, N268D, C275A, C277A, and K292C)^{20 (link)}. The expression and purification of p53 were conducted by following our reported method^{20 (link)}. Briefly, p53 with the GST tag was expressed in Escherichia coli BL21 (DE3) pLysS cells. The cells were lysed by sonication, and the supernatants were loaded onto a GST column (GSTrap FF; GE Healthcare, Tokyo, Japan). The GST tag was cleaved using PreScission Protease (GE Healthcare), and samples were collected. The samples were purified further using a heparin column (HiTrap Heparin HP; GE Healthcare). DNA-binding ability of the purified sample was confirmed using a titration measurement based on fluorescence anisotropy^{8 (link)}. The purified p53 was labeled with ATTO532 using maleimide chemistry, as reported in our previous study^{20 (link)}. The p53 sample labeled with ATTO532 was purified using a cation exchange column (HiTrap SP HP; GE Healthcare). The labeling ratio was determined to be 0.8 dyes/monomer.

The PameOBPs were expressed by using Escherichia coli expression system. To generate properly folded protein, the signal peptide predicted by SignalIP 4.1 [18 (link)] was removed. The two PameOBPs were amplified by gene special primers (S1 Table) with the cDNA as the template. The purified PCR products were ligated into expression vector pGEX-4T-1 using In-Fusion^® HD Cloning Kit (Clontech, Mountain View, CA) according to the manufacturer’s instructions. The recombinant plasmid was transformed into E. coli BL21 (DE3) cells, and then expression and purification was performed according to a previously reported protocol [19 (link)]. Recombinant proteins in the supernatant were purified by an affinity chromatography column GSTrap FF (GE Healthcare, Piscataway, NJ, USA). The GST tags were cleaved from recombinant proteins using 400 μL of thrombin (1U/μL) while loaded on GSTrap FF column at 25°C for 10 h and allowed 12 h. After that, the recombinant proteins were eluted with PBS buffer. Finally, the eluted proteins were desalted using HiTrap Desalting (GE Healthcare), lyophilized, and stored at −80°C until use.

To purify the recombinant CAV VP1 and VP3 proteins, the previously created recombinant E. coli strains BL21 (DE3)-pLysS expressing VP1 and VP3 were used to express recombinant proteins [29 (link)]. The recombinant E. coli cells were cultured, and the harvested cells were disrupted and prepared following a previously described procedure [29 (link)]. Cells were spun down from 50 ml of culture supernatant and resuspended in GST resin binding buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.3). After cell disruption, the resulting cell supernatant was loaded onto a GSTrap FF affinity column (GE healthcare, Piscataway, NJ) for protein purification following the operational conditions described in a previous study [29 (link)]. The total protein concentration of recombinant CAV VP1 and VP3 proteins was determined using a Micro BCA kit (Pierce, Rockford, IL) with bovine serum albumin as the reference protein. Purified VP1 and VP3 proteins were dialyzed against DNA-binding buffer (50 mM Tris-HCl, pH 7.5, 120 mM KCl, 1.0 mM EDTA, 0.5 mM DTT, and 30 mg/ml BSA) and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Purified proteins were stored at − 20 °C until required.

Antibody against BMAL1 was generated as described previously [22] (link). Purified glutathione S-transferase (GST) –Gm129 N-terminal (amino acids 1 to 187) protein was produced as a recombinant protein in the competent cells BL21 (DE3) (Stratagene). After removing GST by using GSTrap FF and PreScision Protease (GE Healthcare), the produced antigen was used to immunize rabbits. The antiserum was subjected to affinity purification using Affi gel 10 (Bio-Rad) conjugated with the antigen. The anti-CHRONO antibody recognized its target protein in immunochemical analysis (Figure S4D).