Escherichia coli bl21 de3

The hKeap1 Kelch domain (residues Ala321–Thr609) and hBcl6 domain (residues Ala5–Glu129) were amplified by PCR using human cDNA libraries. Three cysteine residues of hBcl6 were then mutated (Cys8Gln, Cys67Arg, Cys84Asn) as reported^{52 (link)}. Hereafter, this mutant is referred to as hBcl6. hKeap1 and hBcl6 were ligated into a pET21 vector (Merck Millipore) next to the His-Avi and His-Avi-SUMO-FLAG tags (LifeSensors), respectively. The proteins were expressed with isopropyl β-D-1-thiogalactopyranoside (IPTG) induction in Escherichia coli BL21 (DE3) (Nippon Gene). The proteins were purified using Ni–NTA (FUJIFILM Wako Pure Chemical) and Superdex 200 (GE Healthcare). Next, the purified proteins were enzymatically biotinylated in vitro. Briefly, the proteins were incubated for 3 h at 30 °C with purified Escherichia coli BirA in the presence of D-biotin, magnesium chloride (MgCl₂), and ATP, which was replaced with final buffer (50 mM Tris-hydrochloride [HCl, pH 8.0], 150 mM sodium chloride [NaCl], and 5% glycerol). The proteins were concentrated and quantified using a TaKaRa bicinchoninic acid (BCA) protein assay kit (Takara Bio). The biotinylation rate of the Avi tag was calculated from its protein binding rate to streptavidin sepharose high-performance (GE Healthcare).