Rosetta 2 de3 competent cells

The DBD of Fkh1 and its surrounding residues (amino acids 243–484) were cloned into the pET-28b(+) DNA vector such that a His-Tag is added to the N-terminal end of the polypeptide (Millipore Sigma: 69865). The product was then transformed into Rosetta 2(DE3) Competent Cells (Millipore Sigma: 71405) and expression was induced overnight at 16°C using 0.2 mM IPTG. Cells were centrifuged at 4°C and resuspended in an MCAC-0 buffer (20 mM Tris–Cl pH 8.0, 0.5 M NaCl, 10% glycerol, 1 μg/ml pepstatin A, 50 μg/ml TPCK, 1 mM benzamidine, 1 mM PMSF) on ice. The cells were then broken by sonication and the lysate was clarified by centrifugation at 4°C. The lysate was then incubated with a Ni-NTA resin (Qiagen: 30210) equilibrated in MCAC-0. Next, the resin was sequentially washed with MCAC buffer containing 10, 20, 30, 40 mM imidazole, followed by elution with 250 mM imidazole. Salts were then removed by buffer exchange using Amicon Ultra Centrifugal Filters (Millipore Sigma: UFC901024, UFC800324). The final protein products were verified using SDS-PAGE (Supplementary Figure S4). The same procedure was followed for the expression and purification of the DBDs of Fkh2 (amino acids 280–520), Hcm1 (amino acids 41–213), and Fhl1 (amino acids 401–638), using pET-28a(+) as the DNA vector (Millipore Sigma: 69864).