Hiload 16 600 superdex 75 column

WT spSrtA and saSrtA proteins were expressed and purified as previously described (22 (link)). All other constructs, including chimeric and mutant proteins, were purchased from GenScript in the pET28a(+) vector. In general, protein expression and purification protocols were very similar to those previously described (22 (link)). Briefly, plasmids were transformed into E. coli BL21 (DE3) competent cells and grown in LB media, with protein induction at A₆₀₀ 0.6 to 0.8 using 0.15 M IPTG for 18 to 20 h at 18 °C.
After cell harvest in the lysis buffer [0.05 M Tris, pH 7.5, 0.15 M NaCl, and 0.0005 M EDTA], the protein was purified using a 5-ml HisTrap HP column (GE Life Sciences, now Cytiva), using wash [0.05 M Tris, pH 7.5, 0.15 M NaCl, 0.02 M imidazole, pH 7.5, and 0.001 M TCEP] and elution [wash buffer, with 0.3 M imidazole, pH 7.5] buffers. SEC was conducted using a HiLoad 16/600 Superdex 75 column (GE Life Sciences, now Cytiva) in the SEC running buffer (0.05 M Tris, pH 7.5, 0.15 M NaCl, and 0.001 M TCEP). Purified protein corresponding to the monomeric peak was concentrated using an Amicon Ultra-15 Centrifugal Filter Unit (10,000 NWML) and analyzed by SDS-PAGE and analytical SEC (Figs. S1 and S6). Protein not immediately used was flash-frozen in the SEC running buffer and stored at −80 °C.

ScFv16 was expressed in High-Five™ insect cells (ThermoFisher Scientific, Cat#B85502) as a secreted protein purified by Ni-sepharose chromatography column^{49 (link)}. The HiLoad 16/600 Superdex 75 column (GE Healthcare) was used to separate the monomeric fractions of scFv16 with a running buffer containing 20 mM HEPES and 100 mM NaCl, pH 7.4. The purified scFv16 was flash-frozen in liquid nitrogen with 10% glycerol and stored at −80 °C until use.

Nb35 with a C-terminal histidine tag (His6) was expressed in Escherichia coli BL21 (DE3) bacteria and cultured in Terrific Broth medium supplemented with 2 mM MgCl₂, 0.1% (wt/vol) glucose, and 50 μg/mL ampicillin to an OD₆₀₀ value of 1.0 at 37 °C. The cultures were then induced by 1 mM isopropyl-β-d-thiogalactoside and grown for 5 h at 37 °C. Cells were harvested by centrifugation (4,000 rpm, 20 min), and Nb35 protein was extracted and purified by nickel affinity chromatography as previously described (65 (link)). Eluted protein was concentrated and subjected to a HiLoad 16/600 Superdex 75 column (GE Healthcare) pre-equilibrated with buffer containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.5, and 100 mM NaCl. The monomeric fractions supplemented with 30% (vol/vol) glycerol were flash frozen in liquid nitrogen and stored in −80 °C until use.