Hiload 16 60 superdex 200 pg

HutZ was expressed in Escherichia coli and purified as described previously.³ Briefly, the hutZ gene was subcloned into pET-28b (Merck Millipore, Darmstadt, Germany) via NdeI and EcoRI sites, and the thrombin recognition site (Leu-Val-Pro-Arg-Gly-Ser) in the pET-28b construct was mutated to the HRV 3C protease recognition site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro).^{15 (link)}E. coli strains transduced with HutZ expression plasmids were grown at 37 °C in LB broth supplemented with 50 μg/mL kanamycin. Expression of His-tagged fusion protein in E. coli BL21(DE3) was induced by adding isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.4 mM, after which cells were further incubated at 28 °C overnight. His₆-tagged HutZ was purified by affinity chromatography using a HisTrap HP column (GE Healthcare, Uppsala, Sweden). After cleavage of the His₆-tag, the reaction mixture was applied to the HisTrap column. The column flow-through was collected and then applied to a gel-filtration column (HiLoad 16/60 Superdex 200 pg, GE Healthcare) equilibrated with 50 mM Tris-HCl and 150 mM NaCl (pH 8.0). The protein concentration was determined on the basis of absorbance at 411 nm using extinction coefficient (ε₄₁₂) of 166 mM⁻¹cm^{−1 16 (link)}