Bacterial endotoxin removal beads

For expression of the design variants in E coli, genes were cloned into pET29b between the restriction sites NdeI and XhoI and transformed into BL21Star (Invitrogen). For expression, 10 ml of overnight culture grown in Terrific Broth (BD Difco) was used to start a 500 ml culture using Studier's autoinduction medium but replacing the N-Z-amine fraction with TB. Cells were grown for 8 h at 37°C, before reducing the temperature to 18°C for another 14-16 h. Cells were re-suspended in 35 ml phosphate buffered saline (PBS, 150 mM NaCl and 25 mM phosphate buffer at pH 7.4) and lysed using a M110P Microfluidizer (Microfluidics). Insoluble cell debris was removed by centrifugation for 20 min at 40,000 × g. Supernatant was applied to gravity-flow columns containing 2 mL of Ni-NTA for each 500 ml of culture, washed with 50 ml PBS and 50 ml PBS containing 30 mM imidazole. Proteins were eluted with 20 ml of 250 mM imidazole in PBS. If necessary, proteins concentrated to 1.5 mg/ml using a Vivaspin 10kD MWCO centrifugal concentrator (Sartorius Stedim) at 4000 × g. For animal studies, proteins were purified using column and an imidazole gradient. The 400 mM elution fraction was subjected to further cleaning via SEC and further processed using bacterial endotoxin removal beads (Miltenyi Biotec).