Ultrasonic crasher

Escherichia coli BL21(DE3) cells harboring plasmid pET-P-Intein-ELK16 or pET-Trx-P-Intein-ELK16 were inoculated into Luria–Bertani (LB) medium supplemented with 50 mg/L kanamycin and incubated at 37 °C with shaking (250 rpm). Isopropyl β-D-1-thiogalactopyranoside was added to a final concentration of 0.2 mM to initiate protein expression when OD600 reached 0.4–0.6. The cultures were then continued for an additional 6 h at 30 °C (for the peptide GLP-1 and Ex4, expression was carried out at 37 °C for 6 h to achieve better expression), and then harvested by centrifugation at 6000×g for 10 min and pellets were stored at −70 °C for further assay and analysis.
Harvested cell pellets were re-suspended in buffer B1 (20 mM Tris–HCl, 500 mM NaCl, 1 mM EDTA, pH 8.5) to 10 OD culture/mL, followed by sonication (Ultrasonic crasher; Scientz JY92-IIN, Ningbo, China). The soluble fractions were isolated from the aggregates by centrifugation at 15,000×g for 15 min at 4 °C. The precipitates were washed twice with buffer B1, and re-suspended in the same volume of Buffer B3 (20 mM Tris–HCl, 500 mM NaCl, 1 mM EDTA, 40 mM dithiothreitol, pH 8.5). Intein-mediated cleavage reactions were performed by incubating the samples at 4 °C overnight. Then, the soluble and insoluble fractions were separated by centrifugation at 15,000×g for 15 min at 4 °C.

Harvested cell pellets were re-suspended in buffer B1 (20 mM Tris-HCL, 500 mM NaCl, 1 mM EDTA, pH 8.5) to 10 OD culture/ml, followed by sonication (Ultrasonic crasher, Scientz JY92-IIN, Ningbo, China). The soluble fractions were isolated from the lysates by centrifugation at 11,000 rpm for 10 min at 4 °C. The precipitates were washed twice with buffer B1, resuspended in the same volume of Buffer B3 (20 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA, 40 mM Dithiothreitol, pH 8.5). Intein-mediated cleavage reactions were performed by incubating the samples at 4 °C overnight. Then the soluble and insoluble fractions were separated by centrifugation at 11,000 rpm for 15 min at 4 °C. The protein samples were analyzed by denaturing polyacrylamide gel electrophoresis using precast NuPAGE^® precast 4–12% Bis-Tris gels from Invitrogen, followed by staining with Coomassie Brilliant Blue G-250. The compositions and protein concentrations of all samples were determined densitometrically with Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin (BSA) and aprotinin as standards, and were adjusted according to the loading volume of the protein samples.