Constant cell disruptor system

Single colonies were used to inoculate 50 mL of LB medium supplemented with the corresponding antibiotic. Those overnight cultures were used as starting material to inoculate large volume media (500 mL). All CYPs were grown on TB media supplemented with 15 μM FeCl₃ at 37 °C until the optical density at 600 nm (OD₆₀₀) reached 0.8–1 at which point 500 μM δ-aminolevulinic and 50 mM arabinose were added to support protein expression. After induction, cells were grown for 20–24 hours at 25 °C (CYP101A1 and CYP102A1) or 20 °C (CYP153A6) and collected by centrifugation at 4 °C. Cell pellets were resuspended (200 mg·mL⁻¹) in 50 mM potassium phosphate buffer pH 7, 200 mM NaCl and 10% glycerol. Cells were lysed using a constant cell disruptor system (Constant Systems Ltd, UK). Protease inhibitor tablets (Sigma) were added to the cell lysates to avoid enzymatic degradation of over-expressed proteins. Cell lysates were immediately used for biotransformation or kept frozen at −80 °C. Heme iron thiolate coordination was established by formation of the Fe(II)CO complex at 450 nm after dithionite-reduction and bubbling with carbon monoxide gas. CYPs concentrations were calculated using the extinction coefficient ε₄₅₀ = 96,000, as described previously^{39 (link)}.

GST-tagged constructs were expressed in Rosetta (DE3) Escherichia coli overnight at 18°C. Cells were harvested, resuspended in lysis buffer (20 mM Tris pH 8.5, 500 mM NaCl, 2 mM EDTA), lysed using a Constant Cell Disruptor System (Constant Systems) and spun at 35000 rpm for 20 min at 4°C in a Beckman Ti45 rotor. The cleared supernatant was bound to glutathione beads for 1 h at 4°C. Beads were washed extensively with lysis buffer and resuspended in HEPES buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP). Proteins bound to glutathione beads were used directly for pull downs. Rat brain lysates were incubated with protein-bound beads for 1h at 4°C followed by 6 washes with lysis buffer. Recombinant proteins were incubated with protein-bound beads for 10 min at room temperature and also washed 6 times with lysis buffer. Samples were run on SDS-PAGE gels and either immunoblotted or Coomassie stained. Rat brain lysates were prepared as follows: One brain was defrosted on ice and homogenized in a 15 mL Teflon-glass homogenizer with 4 mL of homogenization buffer (150 mM NaCl, 20 mM HEPES pH 7.5, 2 mM DTT, 1/1000 protease inhibitor cocktail and 0.1% Triton X-100). The lysate was cleared by centrifugation at 50000 rpm in a Beckman TLA 100.4 rotor.