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.