Expression and characterization of recombinant mast cell tryptase.
Tryptase, a serine protease, is the major protein component in mast cells. In an animal model of asthma, tryptase has been established as an important mediator of inflammation and late airway responses induced by antigen challenge. Human tryptase is notable for its tetrameric structure, requirement of heparin for stability, and resistance to endogenous inhibitors. Human protryptase was expressed as a recombinant protein in Pichia pastoris. The recombinant protein consisted of two forms of protryptase, one containing the entire propeptide and the other containing only the Val-Gly dipeptide at its amino terminus. Isolation of active recombinant tryptase required a two column purification protocol and included a heparin- and dipeptidyl peptidase I-dependent activation step. Purified recombinant tryptase migrated as a tetramer on a gel filtration column and displayed kinetic parameters identical to those of a native tryptase obtained from HMC-1 cells, a human mast cell line. Recombinant and HMC-1 tryptase exhibited comparable sensitivities to an array of protein and low-molecular-weight inhibitors, including one that is highly specific for tryptase (APC-1167). Similarly, the recombinant enzyme cleaved both alpha- and beta-chains of fibrinogen to generate fibrinogen fragments indistinguishable from those generated by HMC-1-derived tryptase. Thus, recombinant tryptase expressed in P. pastoris displays physical and enzymatic properties essentially identical to the native enzyme. This system provides a cost-effective and easy to manipulate expression system that will enable the functional characterization of this unique enzyme.
Chan
H
H
Department of Molecular Biology, Department of Enzymology, Department of Pharmacology, Axys Pharmaceuticals, Inc., 180 Kimball Way, South San Francisco, California, 94080, USA. hedy-chan@axyspharm.com
Elrod
K C
KC
Numerof
R P
RP
Sideris
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Clark
J M
JM
D001665:Binding Sites; D053818:Chymases; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010843:Pichia; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D053802:Tryptases
D018076:DNA, Complementary; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases