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Expression and characterization of recombinant mast cell tryptase
H Chan1, K C Elrod, R P Numerof
1Department 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
Protein Expression and Purification
|March 27, 1999
Summary
Recombinant human tryptase, a key mast cell enzyme, was successfully produced in Pichia pastoris. This cost-effective system yields functional tryptase identical to native forms, aiding asthma research.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Tryptase, a serine protease and major mast cell protein, mediates asthma inflammation and airway responses.
- Human tryptase exhibits a tetrameric structure, requires heparin for stability, and resists endogenous inhibitors.
Purpose of the Study:
- To express and characterize recombinant human protryptase in Pichia pastoris.
- To establish a cost-effective and functional system for producing human tryptase for further research.
Main Methods:
- Human protryptase was expressed in Pichia pastoris, yielding two forms.
- A two-column purification and heparin/dipeptidyl peptidase I-dependent activation yielded active recombinant tryptase.
- Characterization involved gel filtration, kinetic analysis, inhibitor sensitivity assays, and fibrinogen cleavage studies.
Main Results:
- Recombinant tryptase purified as a tetramer and showed identical kinetic parameters to native HMC-1 tryptase.
- Both recombinant and native tryptase displayed similar sensitivities to various inhibitors, including a specific tryptase inhibitor (APC-1167).
- Fibrinogen cleavage patterns by recombinant and native tryptase were indistinguishable.
Conclusions:
- Recombinant human tryptase expressed in Pichia pastoris possesses essentially identical physical and enzymatic properties to the native enzyme.
- The Pichia pastoris expression system offers a cost-effective and manageable method for producing functional tryptase.
- This system facilitates the ongoing functional characterization of human tryptase.