Sonoplus ultrasonic homogenizer

Batch cultivation of recombinant E. coli was performed in 3 l Luria–Bertani medium (LB medium, DSM 381) supplemented with 100 μg/ml ampicillin at 37 °C. Recombinant protein production was induced at an optical density of 1 (600 nm) by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Cells were harvested by centrifugation (4 °C, 10.000g, 10 min), re-suspended in 100 mM sodium acetate/citrate/borate buffer (pH 8.5) and disrupted by sonication on ice in 3 cycles in 1 min, 120 W, 50% pulse and 50% power (Sonoplus ultrasonic homogenizer equipped with a UW 2200 ultrasonic head KE76, Bandelin, Berlin, Germany). The γ-CGTase in the soluble crude extract fraction was purified by affinity chromatography with 1 ml His Trap^™ FF Crude Columns (GE Healthcare, Munich, Germany). Purification results were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis [64] (link) following the determination of protein concentration [65] (link).

Briefly, 14.6 g calcium chloride (CaCl₂, Carl Roth, Karlsruhe, Germany), 14.3 g disodium hydrogenphosphate (Na₂HPO₄, Carl Roth, Germany), and 0.8 g sodium fluoride (NaF, Carl Roth, Germany) were mixed in dry state, and 40 mL of MilliQ water were added shortly before ultrasonication [43 ] for 5 min at an energy intake of 18 kJ using a Sonoplus Ultrasonic Homogenizer (Bandelin, Berlin, Germany) and a KE76 probe. Particles were washed with MilliQ water and air dried at 50 °C overnight.
FAp particles were coated in an aqueous solution using eADF4(κ16) dissolved in 6 M guanidinium thiocyanate and dialyzed against 10 mM Tris buffer, pH 7.5 for 16 h. The coating was performed using 10 mg particles in 1 mg/mL protein solution for 8 h, followed by centrifugation at 13,000 rpm for 10 min and washing in MilliQ water. κFAp refers to FAp particles coated with eADF4(κ16).