Pet21 vector

The genes for MmCpnK256C and MmCpnP255C were made by site-directed mutagenesis of the gene of the Cys-less mutant of MmCpn. The MmCpn variants were overproduced in E. coli strain Rosetta (DE3) pLysS using a pET21 vector (Merck Millipore; Billerica, MA, USA). The cells were harvested by centrifugation, suspended in buffer A (50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, and 1 mM dithiothreitol) and disrupted by sonication. The lysate was centrifuged at 15,000× g for 30 min to pellet the cell debris. The supernatant was loaded on a TOYOPEARL SuperQ-650 column (TOYOBO; Osaka, Japan) equilibrated with buffer A. Bound proteins were eluted by a NaCl gradient ranging from 0 to 500 mM. Fractions containing MmCpn variants were pooled and loaded on an AF-Heparin HC-650M (TOYOBO) equilibrated in buffer A with 0.28 M NaCl. Bound proteins were eluted by a NaCl gradient ranging from 0.28 to 1 M. Fractions containing MmCpn variants were pooled and concentrated using an Amicon Ultra-15 10K concentrator (Merck Millipore). Then, the oligomers of MmCpn variants were purified by size exclusion chromatography using a HiLoad 26/60 Superdex200 pg (GE Healthcare, Buckinghamshire, UK) with Buffer B (buffer A + 150 Mm NaCl). Fractions containing the oligomers of MmCpn variants were collected.

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).