Small-scale production of all HheC and HheG mutants was performed in 50 mL reaction tubes in a total volume of 15 mL of Terrific Broth (TB) (per liter: 4 mL glycerol, 12 g peptone, 24 g yeast extract, 0.17 M KH2PO4, 0.74 M K2HPO4) supplemented with 50 µg mL -1 kanamycin and 0.2 mM isopropyl-β-D-thiogalactopyranosid (IPTG), and inoculated with 10% (v/v) preculture. After incubation for 24 h at 22 °C and 220 rpm, cells were harvested by centrifugation (20 min, 3488 g, 4 °C) and the resulting cell pellets were stored at -20 °C until further use. For smallscale purification via N-terminal His-tag, cell pellets were resuspended in 2 mL buffer A (50 mM Tris•SO4, 300 mM Na2SO4, 25 mM imidazole, pH 7.9), supplemented with 1 mg mL -1 lysozyme and one Pierce Protease Inhibitor Mini Tablet (EDTA-free, Life Technologies, Thermo Fisher Scientific). Cells were disrupted by sonication on ice for 3 min (6 cycles of 10 s pulse and 20 s pause). Cell debris were removed by centrifugation (30 min, 21 000 g, 4 °C). The resulting cell-free extracts were loaded on 0.8 mL Pierce® centrifuge columns with a column volume (CV) of 0.6 mL Ni Sepharose TM 6 fast flow (GE Healthcare, Freiburg, Germany), pre-equilibrated with buffer A. After protein binding, columns were washed with each 10 CV of buffer A to remove non-specifically bound proteins. Elution of His6-tagged target proteins was performed using 1.5 CV of buffer B (50 mM Tris•SO4, 300 mM Na2SO4, 500 mM imidazole, pH 7.9) and fractions of each 1 mL were collected. For desalting, 1 mL elution fraction was loaded onto PD MidiTrap TM G-25 desalting columns (GE Healthcare), pre-equilibrated with TE buffer (10 mM Tris•SO4, 4 mM EDTA, pH 7.9, 10% (v/v) glycerol), and eluted with 1.5 mL TE-buffer. Protein concentrations were determined based on absorbance at 280 nm using a NP80 nanophotometer (Implen, München, Germany) and respective molar extinction coefficients obtained by Protparam .