Human cathepsin F: Expression in baculovirus system, characterization and inhibition by protein inhibitors

Recombinant full-length human procathepsin F, produced in the baculovirus expression system, was partially processed during the purification procedure to a form lacking the N-terminal cystatin-like domain and activated with pepsin. Active cathepsin F efficiently hydrolyzed Z-FR-MCA (k_cat/K_m=106 mM^-1s^-1) and Bz-FVR-MCA (k_cat/K_m=8 mM^-1s^-1), whereas hydrolysis of Z-RR-MCA was very slow (k_cat/K_m<0.2 mM^-1s^-1). Cathepsin F was rapidly and tightly inhibited by cystatin C, chicken cystatin and equistatin with K_i values in the subnanomolar range (0.03-0.47 nM), whereas L-kininogen was a less strong inhibitor of the enzyme (K_i=4.7 nM). Stefin A inhibited cathepsin F slowly (k_ass=1.6×10⁵ M^-1s^-1) and with a lower affinity (K_i=25 nM). These data suggest that cathepsin F differs from other related endopeptidases by considerably weaker inhibition by stefins.