Sonifier 250 cell disruptor

2 ml cultivation broth were centrifuged at 20000 g for 10 min at 4℃. The supernatant was discarded and the pellet resuspended in 3 ml 0.1 M NaOH before sonication with a Sonifier®; 250 Cell Disruptor (Branson) (power 70%, duty cycle 40%, power 20 s, pause 40 s, 10 cycles, on ice). The sonicated samples were incubated for 3 h at RT. After centrifugation (20000 g, 10 min, 4℃) the supernatant was used to determine protein concentration with a Bradford assay. Therefore, 20 μl diluted sample (1:10 - 1:100) were added to 1 ml 1:5 diluted Bradford Reagens (Bio-Rad Laboratories) and incubated for exactly 10 min at RT before measuring the absorption on a V-630 UV-Vis spectrophotometer (Jasco, Tokio, Japan) at 595 nm. As standard bovine serum albumin in concentrations from 10 - 100 μg/ml was used.

A total of 300 mL of LB medium with D-glucose (1% w/v) and kanamycin (50 µg/mL) was inoculated with E. coli BL21(DE3)pLysS (Promega, Madison, WI, USA), carrying the respective expression vectors. At OD₆₀₀ protein expression was induced by adding IPTG to a final concentration of 0.5 mM. The culture was incubated at 18 °C for 24 h. The cells were harvested by centrifugation and stored frozen at −20 °C overnight. Cells were then resuspended in a 10 mL binding buffer (0.5 M NaCl, 20 mM Tris-HCl, 5 mM imidazole, pH 7.9) and sonicated using a Sonifier^® 250 Cell Disruptor (Branson, Danbury, CT, USA) (power 40%, duty cycle 70%, power for 30 s, pause for 30 s, 4 cycles). After centrifugation, the protein (105 kDa) was purified from the extract using Novagen^® HisBind^® resin (Merck) and a modified elution buffer (0.5 M NaCl, 20 mM Tris-HCl, 120 mM imidazole, pH 7.9), according to the manufacturer’s guidelines.