Ni nta

The ED3 sequences of DENV1, DENV2, DENV3, and DENV4 serotypes were retrieved from the UniProt database, and the nucleotide sequences, optimized for expression in Escherichia coli, were synthesized and cloned at the NdeI and BamHI sites of pET15b (Novagen) [26 (link)].
All ED3 variants were overexpressed in E. coli JM109(DE3)pLysS as inclusion bodies and refolded as described previously [42 (link)]. In short, after harvesting, the cells were lysed in lysis buffer (150 mM NaCl, 0.5% sodium deoxycholate, and 1% SDS in 50 mM Tris-HCl pH 8.5) and lysis wash buffer (lysis buffer supplemented with 1% v/v NP-40) through sonication. The cell lysates were air oxidized for 36 h at 30 °C in 6 M guanidine hydrochloride in 50 mM Tris-HCl, pH 8.7. The His₆-tagged ED3s were purified by Ni-NTA (Wako, Tokyo, Japan) chromatography, followed by dialysis against 10 mM Tris-HCl, pH 8.0 at 4 °C. The N-terminal His₆-tag was cleaved by thrombin proteolysis [43 (link),44 (link)]. ED3s were purified by a second round of Ni-NTA chromatography followed by reversed-phase (RP) HPLC. The proteins were lyophilized and stocked as powder at −40 °C until use [21 (link)].

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).