TeNT activation
TeNT was activated by cleavage with trypsin to produce the di-chain form, where the light- and heavy-chains are linked only by a disulfide bond. One milligram of purified TeNT was incubated with 200 μL of immobilised TPCK trypsin (Thermofisher) in 0.1 M NH4HCO3, pH 8 at 37°C for three hours as outlined in the manufacturer’s protocol. Immobilised trypsin was separated by centrifugation. Completeness of cleavage was confirmed by SDS-PAGE.