Rosetta de3 competent cells

Recombinant proteins were expressed in Rosetta™ (DE3) competent cells (EMD Millipore, Burlington, MA) in Miller LB supplemented with 100 μg/mL Ampicillin and 34 μg/mL Chloramphenicol at 37 °C until an OD₆₀₀ = 0.6–1.0 was reached; followed by induction with IPTG (1 mM) at 18 °C for 18 h. Cells were harvested by centrifugation and lysed (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 5 mM DTT, Protease inhibitors). GST-tagged protein was immobilized on Pierce® glutathione agarose beads (Thermo Scientific, Waltham, MA) and washed (20 mM Tris-HCl pH 8.0 and 200 mM NaCl). Bound protein was eluted from the beads in wash buffer supplemented with 10 mM glutathione and concentration was determined using Bradford assay. Immobilized protein for crystallography was incubated with TEV at 4 °C for 18 h and subjected to size exclusion chromatography using a Superdex 75 Increase 10/300 GL (GE Healthcare, Chicago, IL) on an AKTA FPLC (GE Healthcare, Chicago, IL) in lysis buffer. The peak 215 nm fractions were collected. SDS-PAGE analysis was employed to determine purity, and protein was flash frozen and stored at −80 °C until needed.

Rosetta™(DE3) competent cells (70954, Merck, Darmstadt, Germany) were transformed with pGEX-6P-1-Ku70-WT, -S77A, -S77D, -S77E, -SS77/78AA, -SS77/78DD, or -SS77/78EE plasmid, respectively, grown until OD600 reached to 0.4, and incubated with 1 mM isopropyl β-d-1-thiogalactopyranoside at 37 °C for 2 h. The cells were collected, suspended in lysis buffer (0.1% Triton X-100, 0.1% Lysozyme, 1 mM DTT, protease inhibitor cocktail (539134, Calbiochem, San Diego, CA, USA)), and sonicated ten times on ice. Glutathione S-transferase (GST)-fusion proteins were purified by glutathione-sepharose 4B (17075601, Cytiva, Marlborough, MA, USA) and eluted with 2 ml of GST elution buffer (10 mM Glutathione, 0.1% Triton X-100 pH 8.0, and protease inhibitor cocktail (539134, Calbiochem, San Diego, CA, USA)).

The recombinant human Drp1 protein (His-tagged human Drp1 isoform 3, kindly provided by Professor Michael Ryan, Monash University, Australia) was generated as described previously^{23 (link)}. Briefly, cDNA for human Drp1 (isoform 3) was cloned into pET21b vector and transformed in Rosetta (DE3) competent cells (Novagen, MA, Merck Millipore). Transformed cells were propagated in Luria–Bertani broth and protein expression was initiated by the addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside. Cells were harvested by centrifugation at 3,500 g for 20 min at 4 °C, and the pellet was re-suspended in lysis buffer containing 50 mM Tris HCl (pH 7.3), 500 mM NaCl, 50 mM imidazole, 2.5 mM β-mercaptoethanol, 0.01 mM LEUPEP, 0.1 mM AEBSF and 1 mM benzaminidium chloride. The cells were lysed using a pre-cooled EmulsiFlex-C5 homogenizer (Avestin, Ontario, Canada) on ice and clarified by centrifugation at 17,000 g for 30 min. The clarified cell lysate was loaded onto a 5 mL Nickel Chelating Sepharose Fast Flow column (GE Healthcare, Buckinghamshire, UK) and Drp1 protein was eluted with wash buffer containing 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 2.5 mM β-mercaptoethanol and 400 mM imidazole. Eluted proteins were equilibrated with a buffer containing 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 10% glycerol and 2 mM TCEP using a PD-10 desalting column (GE Healthcare).