Hiload 16 600 superdex 200 column

pPJ628 (proGeh) and pPJ650 (mGeh) expression vectors were transformed into BL21(DE3) cells and grown in rich broth to an OD₆₀₀ of 0.7 at 37°C with shaking at 200 rpm. The culture was cooled to 16°C and the cells were induced with 1 mM IPTG overnight with shaking at 200 rpm. Cells were harvested and resuspended in 20 mM Tris (pH 8.0), 200 mM NaCl, 10 mM imidazole. Cells were broken by two passages through a cell disruptor and centrifuged at 20,000 g for 45 min. Geh was purified from the supernatant using a Ni-NTA column by washing with 20 column volumes of each 20 mM Tris (pH 8.0), 200 mM NaCl containing 10 mM imidazole or 20 mM imidazole or 40 mM Imidazole. Geh was eluted with 20 mM Tris (pH 8.0), 200 mM NaCl, 250 mM imidazole. The eluant was further purified by gel filtration chromatography using a HiLoad 16/600 Superdex 200 column (Cytiva Life Sciences) equilibrated with 200 mM NaCl, 20 mM Tris, pH 7.5. The molecular weight was estimated by analyzing the peak size on a HiLoad 13/300 Super 200 analytical column (Cytiva Life Sciences). A standard curve was generated using thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), RNase A (13.7 kDa), and aprotinin (6.5 kDa).

RBD probes for detection of RBD-specific B cells were obtained by transfection of Expi293 cells with plasmids expressing each RBD variant using the 293fectin transfection reagent (Thermo Fisher Scientific). At day 5 after transfection, the cell supernatants were clarified by centrifugation and filtered. The RBD probes were purified from the supernatant by HisTrap FF column (Cytiva) and HiLoad 16/600 Superdex 200 column (Cytiva) using the AKTA go (Cytiva). The purified RBD probes were biotinylated using a biotin-protein ligase kit (Avidity, Aurora, CO, USA). For flow cytometric analysis, the biotinylated RBD probes were used^{28 (link)}.

The purified Dicer and TRBP proteins were mixed at a 1:3 molar ratio and loaded onto a HiLoad 16/600 Superdex 200 column (Cytiva) equilibrated with buffer D. The fractions enriched with complex were pooled, concentrated, and flash frozen in liquid nitrogen for use in processing assays.

TPP1-POT1-TIN2 (TPT) purification was carried out previously, as described in Sekne et al. ^{36 (link)}. Briefly, TPT was expressed in Spodoptera frugiperda (Sf9) (Sf9, Oxford Expression Technologies Ltd, Cat# 600100) cells and purified as previously described^{36 (link)}. Briefly, baculoviruses expressing TPP1, POT1, and TIN2 were used to infect 1 L of Sf9 cells, which were grown for 72 h at 27 °C before being harvested by centrifugation. Cell pellets were resuspended in lysis buffer (25 mM HEPES NaOH pH 8.0, 300 mM NaCl, 1 mM MgCl₂, 0.01 mM ZnSO₄, 0.01% IGEPAL CA-630, 1 mM PMSF, 1 mM DTT and 1x cOmplete protease inhibitor tablets (Roche, Cat# 11873580001)). Lysates were sonicated, ultracentrifuged, filtered and bound to dextrin Sepharose resin (Cytiva, Cat# 28-9355-97), followed by an overnight elution by on-column cleavage with SUMOstar protease (LifeSensors, Cat# SP4110). The sample was further purified on a 5 mL HiTrap heparin HP column (Cytiva, Cat# 17040701) and a HiLoad 16/600 Superdex 200 column (Cytiva, Cat# 28-9893-35) in SEC buffer (25 mM HEPES NaOH pH 8.0, 300 mM NaCl, 1 mM MgCl₂, 0.01 mM ZnSO₄, 0.01% IGEPAL CA-630, 10% glycerol, 1 mM PMSF, 1 mM DTT). Peak fractions were pooled, snap-frozen in liquid nitrogen, and stored at −80 °C until use.