Ni smart beads 6ff

Nb35^{45 (link)} with a C-terminal His6 tag was expressed in Escherichia coli BL21 (DE3) and cultured in LB medium with 50 μg/mL ampicillin to an optical density (OD₆₀₀) value of 0.6–1.0 at 37 °C, 180 rpm. IPTG (1 mM) was added to induce Nb35 expression at 27 °C and 180 rpm for 8 h. E. coli cells were then collected by centrifugation (4000 rpm, 20 min) and disrupted in 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol and 1 mM PMSF. Cell pellets were removed by centrifugation (8000 rpm, 30 min), and the supernatant was purified by nickel affinity chromatography (Ni Smart Beads 6FF, Smart Life Sciences). The resin was washed with 30 column volumes of buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 25 mM imidazole and 10% glycerol, and the eluted protein was collected with buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 200 mM imidazole and 10% glycerol. The eluted Nb35 was concentrated and loaded to a HiLoad 16/600 Superdex 75 column (GE Healthcare) preequilibrated with buffer containing 20 mM HEPES, pH 7.4, and 100 mM NaCl. The monomeric fractions were collected and stored with 30% (v/v) glycerol at −80 °C for future use.

Nanobody-35 (Nb35) was expressed and purified similarly as previously described^{26 (link)}. Nb35 was purified by nickel affinity chromatography (Ni Smart Beads 6FF, SMART Lifesciences), followed by size-exclusion chromatography using a HiLoad 16/600 Superdex 75 column and finally spin concentrated to 5 mg/mL.