Emulsiflex c 5 apparatus

The biomass was resuspended by weighing 1 g in 40 mL of PBS buffer pH 7.4. The cells were disrupted by passing eight times through a high-pressure homogenizer cell at 48 MPa (480 bar) using an EmulsiFlex C-5 apparatus (Avestin, Inc). The homogenized product was centrifuged at 4470× g for 20 min, and the cell disruption supernatant was separated from the pellet. The latter was resuspended in the same volume obtained from the rupture supernatant using PBS buffer pH 7.4, and both samples were stored at -20 °C. The Qm1 expression was detected by SDS-PAGE with the denaturing condition and by Western blotting using an anti-poly-histidine monoclonal antibody and anti-Cap polyclonal antibody.

His₆ Rab10-Q63L (1–181), His₆ Mst3, His₆ Myosin Va GTD (1,464–1,855), and His₆ Rab8A Q63L were purified in E. coli BL21 (DE3 pLys) as described by Berndsen et al (2019) (link) and Waschbüsch et al (2020) (link). In brief, bacterial cells were grown at 37°C in Lucia Broth medium and induced at A₆₀₀ nm = 0.6–0.7 by the addition of 0.1 mM isopropyl-1-thio-β-D-galactopyranoside (Gold Biotechnology) and harvested after 18 h at 18°C. The cell pellets were resuspended in ice cold lysis buffer (50 mM Tris, pH 8.0, 10% [vol/vol] glycerol, 250 mM NaCl, 10 mM Imidazole, 5 mM MgCl₂, 0.5 mM DTT, 20 μM GTP, and EDTA-free protease inhibitor cocktail [Roche]), lysed by one passage through an Emulsiflex-C5 apparatus (Avestin) at 10,000 lbs/in², and centrifuged at 13,000 rpm for 25 min in a FiberLite F15 rotor (Thermo Fisher Scientific). Clarified lysate was incubated with cOmplete Ni-NTA resin (Roche) for 2 h at 4°C. Resin was washed three times with five column volumes lysis buffer and eluted in 500 mM imidazole-containing lysis buffer. The eluate was buffer exchanged and further purified by gel filtration on Superdex-75 (GE Healthcare) with 50 mM Hepes, pH 7.5, 5% (vol/vol) glycerol, 150 mM NaCl, 5 mM MgCl₂, 0.1 mM tris(2-carboxyethyl)phosphine (TCEP), and 20 μM GTP.