Microfluidizer

All recombinant proteins were expressed in E. coli BL21(DE3) codon plus and induced by 1.0 mM isopropyl thiogalactoside (IPTG) at 16 °C overnight. The bacterial pellets were lysed using a microfluidizer (Microfluidics) in binding/ATPase buffer (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, and 5% Glycerol) with protease inhibitors (Sigma) and centrifuged at 15,000 × g for 30 min. GST-tagged proteins were purified by using glutathione beads, respectively following manufacturer’s instructions. Protein samples eluted from the beads were further purified by size exclusive chromatography (GE Healthcare) using the ATPase buffer. The purity of the recombinant proteins was checked by Coomassie staining and western immunoblotting.

E.coli BL21 (DE3) cells (New England Biolabs) were grown in TB media until D₆₀₀ of 0.9 and recombinant protein expression was induced with 1mM IPTG for 4hr at 37°C. Both proteins were expressed in insoluble inclusion bodies. E.coli cells were harvested by centrifugation (5000g for 15minutes at 4°C), resuspended in the ice-cold lysis buffer (100mM Tris-HCl pH 7.0, 5mM EDTA, 5mM DTT, 0.5mM PMSF) and lysed by passing through a microfluidizer under 20K psi (Microfluidics). Inclusion bodies were sedimented (50,000g for 20minutes at 4°C) and resuspended using wash buffer (100mM Tris-HCl pH 7.0, 5mM EDTA, 5mM DTT, 2M Urea, 2% w/v Triton X-100). This step was repeated twice and detergent was removed by resuspending inclusion bodies in detergent-free wash buffer (100mM Tris-HCl pH 7.0, 5mM EDTA, 2mM DTT). Finally, the purified inclusion bodies were denatured in 50mM Tris-HCl pH 7.0, 5mM EDTA, 2mM DTT, 6M Guanidine-HCl, protein purity assessed by SDS-PAGE, and quantitated by Bradford assay.

Proteins were expressed using E. coli BL21 (DE3) cells in 2YT media supplemented with either Kanamycin (30 μg/ml) or Ampicillin (100 μg/ml), depending on the plasmid used. Cells were grown to an OD₆₀₀ of 0.8–1.0 at 37°C, then shifted to 18°C. Expression was induced with the addition of 0.4 mM IPTG, and cells were grown further overnight, harvested by centrifugation, and the cell pellets either used immediately for lysis and purification or frozen with LN₂ and stored at −20°C.
All variants of the DENR–MCTS1 complex were purified via an N- or C-terminal His₆-tag using NiNTA and SEC. Cells were resuspended in lysis buffer (30 mM HEPES, 30 mM Imidazol, 500 mM NaCl) and lysed with a Microfluidizer (Microfluidics) at 0.55 MPa. The lysate was cleared by centrifugation for 35 min at 35,000 × g and 4°C, and the resulting supernatant was applied to a 2 ml NiNTA column. The column was washed with 25–50 column volumes of lysis buffer and eluted with elution buffer (lysis buffer plus 400 mM Imidazol). The NiNTA-eluate was applied to a Superdex 200 26/60 column, equilibrated with SEC-buffer I (10 mM HEPES pH 7.5, 500 mM NaCl). Peak fractions containing the DENR–MCTS1 complex were pooled, concentrated to 10–15 mg/ml, and either used directly or shock-frozen with LN₂ and stored at −80°C.

The boosting squalene oil-in-water emulsion was prepared as described previously [40 (link)]. Briefly, a boosting vaccine formulation (200 μL per dose) consisting of rTC_0037 protein (10 μg/dose), immunostimulatory molecule MPLA (1 μg/dose) (Sigma-Aldrich, St. Louis, MO), 50% squalene (Sigma-Aldrich, St. Louis, MO), 0.5% Tween 80, and 0.5% Span 85 in an isotonic phosphate buffer (Sigma-Aldrich, St. Louis, MO) was prepared by homogenization at 12,000 psi with a microfluidizer (Microfluidics, Newton, MA) and passed through a polysulfone filter (220 nm pore size; GE Healthcare, USA) for sterilization. The average diameter (108.3 +/− 8.7 nm) of the emulsion droplets was determined by Nanoparticle Tracking Analysis (NTA) using a NanoSight NS300 (Malvern Ltd., UK).

The plasmid of WT or mutant OPH was transformed into the E. coli strain BL21. The cells were grown in 250 ml standard lysogeny broth (LB) at 37 $${}^{\circ }$$ C and optical density (OD) = 0.4, then induced by 1 mM Isopropyl $$\beta$$ -D-1-thiogalactopyranoside (IPTG) at 16 $${}^{\circ }$$ C overnight. Cells were harvested by centrifugation at 4500×g for 15 min and resuspended in 5 ml of Ni-NTA lysis buffer: 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole and 0.1 $$\mathsf{\mu }$$ M pepstatin at a pH of 7.4. The cells were lysed using Microfluidizer (Microfluidics, Westwood, MA, USA) and then centrifuged at 12,000× g for 30 min. The collected supernatant was incubated with 2 ml of Ni-NTA resin under end-to-end shaking and loaded on a 10 mL HisTrap FF column. After washing with buffer containing 50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, the proteins were eluted with buffer containing 50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole.

Myo19 constructs (Fig. 1A), based on the canonical human isoform (Q96H55-1), containing an N-terminal FLAG tag (DYKDDDDK), the MD, the LC-binding IQ region, and a C-terminal biotin tag (27 (link)), were expressed and purified with CaM and RLC12B (O14950-1) as indicated using methods established previously for class-I myosins (49 (link), 50 (link), 51 (link)).
RLC12B with an N-terminal MBP-tobacco etch virus (TEV) protease cleavage site moiety was expressed in Rosetta(DE3) cells, which were grown in Terrific broth with antibiotic selection to an absorbance of 1.5 and induced overnight with 1 mM IPTG at 18 °C. Cells harvested by centrifugation (4000g for 20 min at 4 °C) were resuspended in ice-cold lysis B buffer (20 mM Tris–HCl [Ph 8], 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 10 μM leupeptin, 1 mM PMSF, and 0.4 mg mL⁻¹ benzamidine) and lysed using a microfluidizer (Microfluidics). Lysates were clarified by centrifugation at 48,000g at 4 °C for 20 min. Proteins were bound to amylose resin, washed, and RLC was eluted by overnight cleavage with TEV protease (4 °C) and dialyzed for 4 h into Buffer-N (20 mM Tris–HCl [pH 8], 200 mM NaCl, and 10 mM imidazole). The protease was removed by binding nickel resin, and the unbound RLC was then dialyzed into Storage Buffer R (20 mM Tris–HCl [pH 8], 100 mM NaCl, 1 mM EGTA, 50% glycerol, and 2 mM DTT) and stored at −20 °C.