Ms 72 probe

Samples of 10 mL DES containing 0.5 M vinyldecanoate were sonicated for 5 min with 60% amplitude and a cycle of 20 s pulsing and 30 s pause in 50 mL tubes. A Sonopuls HD 3100 ultrasonic homogenizer from Bandelin (Berlin, Germany) equipped with a MS 72 probe was used at a frequency of 20 kHz (with an energy input of 4.654 kJ). The probe was set to an immersion depth of 1.5 cm. During sonication, the samples were cooled in a water bath after which they were immediately used for synthesis.

For each plasmid transformed, a recombinant colony was transferred from the agar plate to 20 ml of LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) completed with 100 μg/ml of ampicillin and incubated overnight at 37°C, 180 rpm. The culture was diluted at an OD₆₀₀ value of 0.08 with 200 ml of LB, supplemented with the same antibiotic, and further incubated at 37°C. To induce the recombinant protein expression, 1 mM IPTG (isopropyl-β-d-thiogalacto-pyranoside) was added when the culture reached an OD₆₀₀ value of 0.5. After 3 h of induction, cells were collected by centrifugation at 3,300 × g at 4°C for 15 min. The obtained pellet was resuspended in a lysis buffer (100 mM Tris HCl, 10 mM EDTA, 2 M urea, and 2% Triton X-100 pH 8.0), at a final concentration of 20 OD/ml, and subjected to a sonication process (Bandelin, HD3200, MS 72 probe, running at 40% amplitude) for 30 min (30″ ON and 30″ OFF) in an ice bath. The inclusion bodies were collected by a centrifugation step at 3,300 × g for 15 min and then washed three times with the lysis buffer to remove contaminants. Next, the pellets of the inclusion bodies were dissolved in a denaturing buffer (100 mM Tris HCl, 10 mM EDTA, 8 M urea, and 10 mM DTT, pH 8) and incubated for 1 h at 37°C under stirring. The supernatants were collected after centrifugation at 3,300 × g at 4°C for 15 min.