TAILIEUCHUNG - Báo cáo khoa học: 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 framewith yeasta-factor cDNAand expressedinthePichiapastorisyeast expressionsystem. | Eur. J. Biochem. 271 2370-2378 2004 FEBS 2004 doi Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre-elafin trappin-2 expressed in Pichia pastoris Marie-Louise Zani 1 Shila M. Nobar2. Sandrine A Lacour1 Soazia Lemoine3 Christian Boudier2 . B. Joseph G. Bieth2 and Thierry Moreau1 1INSERM U618 University Francois Rabelais Tours France 2Laboratory of Enzymology INSERM U392 University Louis Pasteur Faculty of Pharmacy Illkirch France 3Laboratory of Marine Biology Universite Antilles-Guyane Campus de Fouillole Pointe a Pitre Guadeloupe France 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. A cDNA coning either elaiin or its precursor. trappin-2 was fused in frame with yeast a-factor cDNA and expressedinthe Pichiapastoris 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-tenninal 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 X 106 M-1-s-1 while dissociation rate constants kdiss were found to be in the 10 4 s 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 .

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