Ni nta resin

The cDNA encoding the BTN2A1 B30.2 domain was cloned into the pET28a vector with an N-terminal or C-terminal 6×His tag. Overexpression of the BTN2A1 B30.2 domain was induced in Escherichia coli BL21 (DE3) cells by 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) (Solarbio) at 18 °C for 24 h. Cells were collected by centrifuging, resuspended and lysed by sonication, and centrifuged at 20,000g for 1 h. The supernatant was collected and incubated with Ni-NTA resin (GE Healthcare), and was then eluted with a buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl and 500 mM imidazole. The eluent was incubated overnight with TEV protease for 6×His tag cleavage, purified by Ni-NTA resin or further purified by DEAE Sepharose chromatography (GE Healthcare). Finally, the protein was dialysed against a storage buffer containing 20 mM HEPES pH 7.5 and 150 mM NaCl. The purified proteins were monitored at all stages of the purification process using SDS–PAGE and visualized by Coomassie blue staining.
All BTN2A1 B30.2-domain mutants were generated using a standard PCR mutagenesis strategy, overexpressed and purified in the same manner as for the WT protein.

Plasmid pET22b-odsR was transformed into E.coli BL-21 DE3 cells. A single colony was inoculated in LB medium overnight at 37 °C. A total of 5 mL of the culture was added into 500 mL autoinduction medium^{33 (link)}. The cells were grown at 37 °C for 4 h, followed by overnight induction at 18 °C. The cells were harvested and cell pellets were resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole, 0.5 mM TCEP, pH 8.0) with 1 mg mL⁻¹ lysozyme and 1 tablet of SIGMAFAST protease inhibitor (Sigma-Aldrich). The cell suspension was sonicated on ice and centrifuged at 15,000 rpm for 1 h. The supernatant was mixed with 1 mL Ni-NTA resin (GE Healthcare Life Sciences) and shaken for 2 h. The Ni-NTA resin was spun down and loaded onto an empty column, which was washed successively with lysis buffer and lysis buffer containing 40 mM imidazole and 60 mM imidazole. OdsR-His protein was eluted in 400 mM imidazole prepared in lysis buffer and further purified by size-exclusion chromatography (SEC) using a Superdex 200 column (16/600, GE Healthcare Life Sciences) in the SEC buffer (20 mM Tris, 150 mM NaCl, 1 mM TCEP, pH 8.0). The fractions containing Ods-His protein were pooled and concentrated to about 8 mg mL⁻¹. The protein solution was aliquot and stocked at −80 °C freezer.

Plasmids encoding the heavy chain and light chain of Abs were transiently co-transfected in pairs, at equivalent molar ratios, into cultured mammalian human embryonic kidney HEK293F cells in Freestyle 293F medium (Invitrogen, CA, USA, 12338018) following the standard protocol [34 (link),35 (link)]. Culture supernatants were collected after 6 days by centrifugation and filtration (0.22 μm, polyethersulfone; Corning). Abs were purified from the culture supernatants using a CaptivA™ Protein A-agarose chromatographic column (Repligen, MA, USA) and were extensively dialyzed to achieve the final composition of phosphate-buffered saline (PBS; pH 7.4). Likewise, the plasmid encoding the pMHC SCT protein was transfected into HEK293F cells. The pMHC protein was purified from the culture supernatant using Ni-NTA resin (GE Healthcare, IL, USA). Protein concentrations were determined using a bicinchoninic acid kit (Thermo Fisher Scientific, Waltham, MA, USA). To prepare an Ab-screening antigen, the purified pMHC SCT proteins were biotinylated using a BirA500 kit (Avidity LLC, Colorado, USA) following the manufacturer’s instructions [35 (link)].