Ja 25.50 fixed angle rotor

Selected CaMKIIβ isoforms were expressed in High Five insect cells via the baculovirus system. All purification steps were performed at 4°C. Cell pellets were resuspended in CaMKII lysis buffer (10 mM Tris–HCl, pH 7.5, 500 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5% glycerol, and 1 mM DTT) supplemented with protease inhibitors (cOmplete; Roche) and lysed by sonication. Insoluble particles were separated by centrifugation at 21,500 rpm for 1 h (JA-25.50 fixed-angle rotor; Beckman Coulter). The soluble fraction was incubated with Strep-Tactin Sepharose beads (IBA Lifesciences) for 1 h and washed with CaMKII lysis buffer. Bound protein was eluted with CaMKII SEC buffer (50 mM Pipes, pH 7.5, 500 mM NaCl, 1 mM EGTA, 10% glycerol, and 1 mM DTT) containing 2.5 mM desthiobiotin (IBA Lifesciences). Eluted protein was concentrated and run on a Superose 6 10/300 Gl size-exclusion column (Cytiva) with CaMKII SEC buffer. Fractions were pooled according to SDS–PAGE and chromatogram, concentrated to ∼1 mg/ml, and flash-frozen in single-use aliquots in liquid nitrogen. Before use, aliquots were thawed on ice, gently mixed by pipetting, and centrifuged at 20,000 rcf for 5 min. Exactly equal concentrations were determined by repeated SDS–PAGE, Coomassie staining, and quantification with ImageQuant TL (Cytiva).

Cells were cultivated on 50 mM glycine betaine in 0.5 to 2 l carbonate buffered complex medium to late exponential growth phase (OD₆₀₀ of 0.3) and then harvested by centrifugation (Avanti J-25 and JA-10 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States) at 8000 rpm and 4 °C for 10 min. The harvested cells were subsequently washed with 30 ml of buffer containing 50 mM imidazole (pH 7.0), 20 mM KCl, 20 mM MgSO₄, 4 mM DTE and 4 µM resazurin by centrifugation at 8500 rpm and 4 °C for 10 min (Avanti J-25 and JA-25.50 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States) and resuspended in 5 ml of imidazole buffer and kept in 16-ml Hungate tubes. All the steps were performed under strict anoxic conditions in an anoxic chamber (Coy Laboratory Products, Grass Lake, MI, United States) filled with N₂/H₂ (96–98%/2–4%; v/v). The total protein concentration in the resting cells was determined according to a previously described method [38 (link)].

A 500 ml cultures of T. kivui were grown in complex or defined medium to late exponential growth phase (OD₆₀₀ of 1.7 to 2.3) and then harvested by centrifugation (Avanti^TMJ-25 and JA-10 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States) at 7,000 × g and 4°C for 10 min. The harvested cells were washed with 30 ml of the respective medium by centrifugation at 8,500 rpm (5948 × g) and 4°C for 10 min (Avanti^TMJ-25 and JA-25.50 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States). Then, the cells were resuspended in 5 ml of the respective medium and kept in 16 ml Hungate tubes. Resuspended cells were distributed into in Hungate tubes to a final volume of 10 mL and a final protein concentration of 10 mg ml^–1. All the steps were performed under strictly oxygen free conditions in an anoxic chamber (Coy Laboratory Products, Grass Lake, MI, United States) filled with N₂/CO₂ (80/20; v/v) for carbonate medium or with 100% N₂ for carbonate free medium. As substrate, 25 mM glucose or 25 mM mannitol was added to the resting cells. The experiment started with incubation at 65°C in water bath with shaking (150 rpm). 0.8 ml subsamples were taken for determination of protein, substrate and product concentration. The total protein concentration in the cell suspension was measured using the method by Schmidt et al. (1963) (link).

The sequence for Syntide 2 (PLARTLSVAGLPGKK) was expressed as a GST fusion protein, with a TEV-cleavable N-terminal His-tag. A short linker (GGGGSGGGGS) was inserted between the Syntide 2 sequence and the C-terminal GST-tag. The fusion protein was expressed in BL21 RIL cells using autoinduction medium. All purification steps were performed at 4°C. Cell pellets were resuspended in lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 20 mM imidazole, and 1 mM DTT) containing protease inhibitors (cOmplete; Roche) and lysed by sonication. Insoluble particles were separated by centrifugation at 21,500 rpm for 1 h (JA-25.50 fixed-angle rotor; Beckman Coulter). The soluble fraction was loaded on a HisTrap Crude column (Cytiva) and eluted with a linear gradient of elution buffer (20 mM Tris–HCl, pH 7.5, 300 mM NaCl, 500 mM imidazole, and 1 mM DTT). Target fractions were pooled, supplied with TEV protease (self-made), and dialyzed against lysis buffer overnight. Digested samples were rerun on a HisTrap Crude column. The flow-through was collected, concentrated, and run on a HiLoad Superdex 75 26/60 size-exclusion column (Cytiva), equilibrated with SEC buffer (20 mM Pipes, pH 7.5, and 50 mM NaCl). Target fractions were pooled, concentrated to 22 mg/ml, and flash-frozen in liquid nitrogen.