α lactose

The following growth media were used: 2xTY medium (20 g/L Tryptone (Casitose Type-I, HiMedia, Mumbai, India), 10 g/L Yeast Extract (HiMedia, Mumbai, India), 20 g/L NaCl (Fisher Scientific, Loughborough, UK), pH 7.2) containing 100 μg/mL Ampicillin Ampicillin (Fisher Bioreagents, Pittsburg, PA, USA) and supplemented with 1 mM MgSO₄ (Fisher Scientific, Loughborough, UK) and 1% Glucose (Merck KGaA, Gernsheim, Germany) (2xTY-Amp); LB medium (10 g/L Tryptone, 5 g/L Yeast Extract, 10 g/L NaCl, pH 7.2) containing 100 μg/mL Ampicillin and supplemented with 1 mM MgSO₄ and 1% Glucose (LB-Amp); ZYP-5052 medium for autoinduction (1% Tryptone, 0.5% Yeast Extract, 25 mM (NH₄)₂SO₄ (Chem-Lab NV, Zedelgem, Belgium₎, 50 mM KH₂PO₄ (Fisher Bioreagents, UK₎, 50 mM Na₂HPO₄ (Fisher Bioreagents, Loughborough, UK), 0.5% glycerol (Sigma, St. Louis, MO, USA), 0.05% Glucose, 0.2% α-lactose (Carl Roth GmbH, Karlsruhe, Germany), 1 mM MgSO₄), containing 100 μg/mL Ampicillin; TYE agar (10 g/l Tryptone, 5 g/l yeast, 8 g/l NaCl, 15 g/l agar), containing100 μg/mL Ampicillin (TYE-Amp).

The different CheF homologs, GST-MmCheF constructs, MmCheY and MmCheY:CheF_CTD were produced in E. coli BL21 (DE3) (Novagen). The protein production was performed in auto-inductive Luria-Miller broth (Roth) containing 1 % (w/v) α-lactose (Roth). The cells were grown for 20 h at 30 °C and 180 rpm. The cultures were harvested by centrifugation (4,000 g, 15 min, 4 °C), resuspended in HEPES buffer (20 mM HEPES, 200 mM NaCl, 20 mM KCl, 40 mM imidazole, pH 8.0), and subsequently disrupted using a microfluidizer (M110-L, Microfluidics). The cell debris was removed by centrifugation (50,000×g, 20 min, 4 °C). The supernatant was loaded onto Ni-NTA FF-HisTrap columns (GE Healthcare) for affinity purification via the hexahistidine tag. The columns were washed with HEPES buffer (10x column volume) and eluted with HEPES buffer containing 250 mM imidazole. The protein was concentrated with Amicon Ultra-30K centrifugal filters and subjected to SEC using a HiLoad 26/600 Superdex 200 column equilibrated in HEPES buffer without imidazole and a pH of 7.5. The peak fractions were analyzed using a standard SDS-PAGE protocol, pooled, and concentrated with Amicon Ultra-30K centrifugal filters.