30 kda spin filters

C-terminally (His)₆-tagged LukE was expressed in BL21 (DE3) Escherichia coli cells (NEB). Transformed cells were grown at 37°C in Terrific broth supplemented with 100 µg/ml ampicillin to a density of OD600 = 0.6. Expression was then induced overnight at 18°C by addition of 0.5 mM IPTG. Cells were harvested by centrifugation (3,000 rpm) and cell pellets were stored at – 80°C until purification. After thawing the frozen cell pellets, cells were lysed by sonication in a lysis buffer consisting of 10 mM Tris (pH 8) and 150 mM NaCl. Lysed cells were centrifuged (16,000 rpm) and the supernatant was loaded onto a nickel NTA Agarose resin. The resin was washed with 20 CV of wash buffer 1 consisting of 50 mM HEPES (pH 7.5) and 1 M NaCl, and with 10 CV of wash buffer 2 consisting of 50 mM HEPES (pH 7.5), 150 mM NaCl and 40 mM imidazole. Bound toxin was eluted with wash buffer 2 supplemented with 200 mM imidazole. The eluted proteins were concentrated using 30 kDa spin filters (Millipore) and further purified by size exclusion chromatography on a Superdex 200 Increase 10/300 column (GE Healthcare) in 50 mM HEPES (pH 7.5) and 150 mM NaCl. Monodisperse elution fractions were pooled and further concentrated prior to crystallization, SAXS and/or cell-based assays.

EOC cell pellets were lysed with RIPA buffer and 100 μg of protein was digested using the FASP method⁴⁴ on 30 kDa spin filters (Millipore). The eluted peptides were acidified and desalted using in‐house made C18 pipette tips (10 μg capacity). Analysis was performed on an Easy nLC‐1200 coupled to a ThermoQExactive HF mass spectrometer (Thermo Fisher Scientific) operating in a top 20 mode. The mobile phase was composed of Buffer A (0.1% formic acid) and Buffer B (0.1% formic acid in 80% acetonitrile). Peptides were separated using a PepMap RSLC C18 2 μm, 75 μm × 50 cm column and a PepMap 100 C18 3 μm, 75 μm × 2 cm precolumn with a 2 hour gradient of 5% to 40% Buffer B. Data were analyzed using MaxQuant (v1.6.10.43)⁴⁵ and Perseus.⁴⁶