Generation of recombinant human tryptase ϵ in COS-7 and insect cells, and spontaneous activation of the recombinant protease. (A) Wild-type pro–tryptase ϵ and its activation-resistant R-1A mutant were expressed in COS-7 cells. After 24 and 48 hours, aliquots of the conditioned medium were subjected to SDS-PAGE and the resulting protein blots were probed with anti–tryptase ϵ antibody (top) or anti-V5 antibody (bottom). (B) Wild-type, COS-7 cell–derived tryptase ϵ (▵, ▴), and its R-1A mutant (▪, □) that had incubated for 24 (▵, □)or 48 (▴, ▪) hours also were evaluated for their enzymatic activity using the tryptase ϵ–susceptible synthetic substrate H-D-Leu-Thr-Arg-pNA. OD indicates optical density. (C-D) Recombinant pro–tryptase ϵ, its C90A mutant, and its C90A/D92E double mutant also were generated using a baculovirus-High 5 insect cell expression system. Insect cells were cultured for 4 days and the resulting condition medium was applied to an anti-FLAG column. Purified wild-type tryptase ϵ and its mutants were incubated at 37°C (C) or 4°C (D) for the indicated times. Aliquots of the resulting products were then evaluated for their enzymatic activity using H-D-Leu-Thr-Arg-pNA (top rows). Other aliquots were subjected to SDS-PAGE under reducing conditions. In the bottom row of panel C, the resulting gels were stained with Coomassie blue. Molecular-mass standards are indicated on the left. In the lower rows of panel D, immunoblots were prepared and probed with anti–tryptase ϵ or anti-FLAG antibodies. Error bars in this and subsequent figures indicate the mean ± standard error of the mean from a single experiment carried out in triplicate. Experiments were carried out more than 2 times to confirm the data.