Nb35 His6 Protein Expression and Purification

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.