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doi:10.1016/j.pep.2006.04.018 
Copyright © 2006 Elsevier Inc. All rights reserved.
Expression and characterization of recombinant γ-tryptase
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Jing Yuana, Jeri Beltmanb, 1, Erik Gjerstadb, Margaret T. Nguyenb, Jun Sampanga, Hedy Chana, 
, 
, James W. Jancb and James M. Clarka
Received 5 January 2006;
Abstract
Tryptases are trypsin-like serine proteases whose expression is restricted to cells of hematopoietic origin, notably mast cells. γ-Tryptase, a recently described member of the family also known as transmembrane tryptase (TMT), is a membrane-bound serine protease found in the secretory granules or on the surface of degranulated mast cells. The 321 amino acid protein contains an 18 amino acid propeptide linked to the catalytic domain (cd), followed by a single-span transmembrane domain. γ-Tryptase is distinguished from other human mast cell tryptases by the presence of two unique cysteine residues, Cys26 and Cys145, that are predicted to form an intra-molecular disulfide bond linking the propeptide to the catalytic domain to form the mature, membrane-anchored two-chain enzyme. We expressed γ-tryptase as either a soluble, single-chain enzyme with a C-terminal His tag (cd γ-tryptase) or as a soluble pseudozymogen activated by enterokinase cleavage to form a two-chain protein with an N-terminal His tag (tc γ-tryptase). Both recombinant proteins were expressed at high levels in Pichia pastoris and purified by affinity chromatography. The two forms of γ-tryptase exhibit comparable kinetic parameters, indicating the propeptide does not contribute significantly to the substrate affinity or activity of the protease. Substrate and inhibitor library screening indicate that γ-tryptase possesses a substrate preference and inhibitor profile distinct from that of β-tryptase. Although the role of γ-tryptase in mast cell function is unknown, our results suggest that it is likely to be distinct from that of β-tryptase.
Keywords: Human γ-tryptase; Human transmembrane tryptase; Mast cell γ-tryptase; Active γ-tryptase
Article Outline
- Materials and methods
- Cloning of human γ-tryptase
- Generation of expression constructs for tc γ-tryptase and cd γ-tryptase
- Expression of recombinant γ-tryptases
- Fermentation and purification of recombinant γ-tryptase
- Deglycosylation of tc γ-tryptase
- Enzyme inhibition assays
- Substrate library screen
- Compound library screen
- Results and discussion
- Purification and characterization of cd γ-tryptase and tc γ-tryptase
- Substrate library screening of cd γ-tryptase
- Inhibitor screening of cd γ-tryptase
- Acknowledgements
- References
