Amicon

T158c was cultivated in complex JG medium [0.75% yeast extract, 0.5% peptone, 0.5 × 10^-3% FeCl₃, 0.5 × 10^-3% MnSO₄, 0.5% MgSO₄, 0.4% (NH₄)₂SO₄, 0.08% KCl, 1.0% glucose, 5.2% glycerol] adjusted to pH 4.7 with McIlvaine buffer (Flegelova et al., 2002 (link)) (adapted). After cultivation for 5 days at 20°C, yeast cells were harvested via centrifugation, and the resulting cell-free supernatant was concentrated by an ultrafiltration device (Amicon, Sartorius) using appropriate ultrafiltration discs (PLCC07610, Merck-Millipore) with a molecular cut-off of 5 kDa. Finally, the toxin concentrate was dialyzed against 10 mM McIlvaine buffer (pH 4.7) over 3 days.

The TrkB cleavage assay was done as described above and shown in the Supplementary Table S4. Only the total volume has been up-scaled to 500 µL.
Processed TrkB ECD was injected into the AKTA FPLC system (GE Healthcare, Vienna, Austria) with a Superdex 200 10/300 GL column (Cytiva Lifesciences) attached, while for TrkB Ig2, a Superdex 75 10/300 GL was used. Both columns were equilibrated with 1x PBS pH 7.4. Fractions with no visible legumain on SDS-PAGE were pooled and concentrated with a 5 kDa cutoff Amicon (Sartorius, Göttingen, Germany).