Reconstitution of M-TC using isolated BoNT and rNTNHA. Reconstitution of the M-TC was achieved by mixing isolated D-4947 BoNT and purified rNTNHA at a molar ratio of 1:1 in 50 mM phosphate buffer (pH 6.0) containing 0.15 M NaCl. After incubation at 25 °C for 2 days, the mixture was applied to a Superdex 200 HR 10/30 gel LY2157299 column equilibrated with the same buffer. The reconstituted complex eluted as a separate peak and was subjected to SDS–PAGE analysis.
Long-term incubation of rNTNHA. The purified rNTNHA (250 μg/ml) was incubated at 25 °C in the dark, and 15 μl aliquots were removed at 2, 4, 7 and 12 days, added to a half volume of SDS–PAGE sample treatment buffer, and subjected to SDS–PAGE. The protease inhibitors 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM EDTA were added to some incubation mixtures. The rNTNHA was quantified by measuring the intensity of the CBB-stained SDS–PAGE band using Image J 1.38v software (http://rsb.info.nih.gov/ij).
Proteolysis with trypsin and pepsin. Solutions of rNTNHA, BoNT and the rNTNHA/BoNT complex (each 1 mg protein) were incubated at 37 °C with 100 μg of TPCK-trypsin (12,500 U/mg) in 50 mM phosphate buffer (pH 6.0) containing 0.15 M NaCl and with 10 μg of pepsin (3100 U/mg) in 50 mM acetate–HCl buffer (pH 2.7), each in a total volume of 1 ml. The reactions were terminated by removal of aliquots from the mixture at appropriate intervals, boiling and subjecting them to SDS–PAGE.