Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre-elafin (trappin-2) expressed in Pichia pastoris

Elafin and its precursor, trappin-2 or pre-elafin, are specific endogenous inhibitors of human neutrophil elastase and proteinase 3 but not of cathepsin G. Both inhibitors belong, together with secretory leukocyte protease inhibitor, to the chelonianin family of canonical protease inhibitors of serine proteases. AcDNAcoding either elafin or its precursor, trappin-2, was fused in frame with yeast a-factor cDNAand expressed in thePichia pastoris yeast expression system. Fulllength elafin or full-length trappin-2 were secreted into the culture medium with high yield, indicating correct processing of the fusion proteins by the yeast KEX2 signal peptidase. Both recombinant inhibitors were purified to homogeneity from concentrated culture medium by one-step cationic exchange chromatography and characterized by N-terminal amino acid sequencing, Western blot and kinetic studies. Both recombinant elafin and trappin-2 were found to be fastacting inhibitors of pancreatic elastase, neutrophil elastase and proteinase 3 with kass values of 2–4 · 10 6 M )1 AEs )1 , while dissociation rate constants kdiss were found to be in the 10 )4 s )1 range, indicating low reversibility of the complexes. The equilibrium dissociation constant Ki for the interaction of both recombinant inhibitors with their target enzymes was either directly measured for pancreatic elastase or calculated from kass and kdiss values for neutrophil elastase and proteinase 3. Ki values were found to be in the 10 )10 molar range and virtually identical for both inhibitors. Based on the kinetic parameters determined here, it may be concluded that both recombinant elafin and trappin-2 may act as potent anti-inflammatory molecules and may be of therapeutic potential in the treatment of various inflammatory lung diseases.