rMaj-pCHH-B

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	The transformation and culture of E. coli strain BL21(DE3) STAR (Thermo Fisher Scientific) with expression plasmids, induction of recombinant protein overexpression, and preparation of the soluble fraction of the cell lysate were performed as per previously described methods (62, 63).

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The transformation and culture of E. coli strain BL21(DE3) STAR (Thermo Fisher Scientific) with expression plasmids, induction of recombinant protein overexpression, and preparation of the soluble fraction of the cell lysate were performed as per previously described methods (62, 63).

Publication protocol

"The transformation and culture of E. coli strain BL21(DE3) STAR (Thermo Fisher Scientific) with expression plasmids, induction of recombinant protein overexpression, and preparation of the soluble fraction of the cell lysate were performed as per previously described methods (62, 63).

The soluble fraction of the cell lysate containing the recombinant fusion protein, His-Nus-His-tagged rMaj-pCHH-B, was purified using the Ni Sepharose 6 Fast Flow resin (GE Healthcare). While the recombinant fusion protein was captured with the resin, the affinity tags were cleaved from rMaj-pCHH-B by protease digestion in a buffer containing 25 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 0.4 M urea, and ProTEV protease (Promega) at 20°C for 24 h. The untagged rMaj-pCHH-B was washed out from the resin and further purified by reverse phase high-performance liquid chromatography (RP-HPLC) on a Capcell Pak C18 SG300 column (150 × 6 mm; Shiseido, Tokyo, Japan) using the following program: a 2-min hold at 5% acetonitrile (MeCN) in 0.05% trifluoroacetic acid (TFA), a 5-min linear gradient of 5–25% MeCN in 0.05% TFA, a 15-min gradient of 29– 37% MeCN in 0.05% TFA, a 1.2-min gradient of 37– 85% MeCN in 0.05% TFA, and a 5-min hold at 85% MeCN in 0.05% TFA at a flow rate of 0.8 mL/min.

The soluble fraction containing recombinant His-Nus-His-tagged rMaj-NPLP was incubated with the Ni Sepharose 6 resin at 4°C for 20 h. The resin was then washed with washing buffer (20 mM phosphate buffer, 0.2 M NaCl, 50 mM imidazole, pH 7.4) and equilibrated with washing buffer without imidazole. For the cleavage of the tags, HRV 3C protease (Takara Bio) was added, and the resin slurry was incubated at 4°C for 3 days. Untagged rMaj-NPLP was eluted from the resin and further purified by RP-HPLC on the aforementioned column using the following program: a 1-min hold at 5% MeCN in 0.05% TFA, a 4-min linear gradient of 5–21% MeCN in 0.05% TFA, a 15-min gradient of 21– 29% MeCN in 0.05% TFA, a 3.25-min gradient of 29–85% MeCN in 0.05% TFA, and a 5-min hold at 85% MeCN in 0.05% TFA at a flow rate of 0.8 mL/min."

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