Skin βI Tryptase, Human, Recombinant (rhSkin β Tryptase) and Lung βII Tryptase, Human, Recombinant (rhLung β Tryptase) are neutral serine proteases. The human β tryptase enzymes have been cloned and stably expressed in Pichia pastoris as fully active tetrameric enzymes and purified by affinity chromatography. The two enzymes differ in buffer formulation, enzyme concentration and glycosylation pattern. rhSkin Tryptase is offered at a concentration of 200µg/ml, while rhLung Tryptase is provided at a much higher concentration (2mg/ml) in minimal buffer without heparin for chromatographic studies and with glycosylation more closely resembling cadaveric enzyme as demonstrated by glycosidase digestion followed by Western analysis of the two recombinant enzymes and native lung tryptase.
Specific activity is measured as the rate of hydrolysis of 0.4mM Nα CBZ-L-Lysine Thiobenzyl Ester as substrate coupled with Ellman's Reagent (5,5´-Dithio-bis(5-Nitrobenzoic Acid)) in a final volume of 1ml, incubating for 1 minute at 25°C, and monitoring the absorbance change at 410nm. One unit is defined as 1.0 absorbance unit change per minute. The specific activity of rhSkin β Tryptase is >1,000 units/mg protein and rhLung β Tryptase is >1,200 units/mg protein.
- Specific activity is consistently 130–150% higher than native lung tryptase.
- Recombinant protein expression results in consistent enzyme from batch to batch.
- Void of human pathogens associated with native cadaveric tryptase.
- Skin β and Lung β Tryptase are free of other contaminating proteases, providing more active enzyme per milligram of protein and eliminating extraneous protein interactions observed with native tryptase.
Tryptase is the predominant protein in mast cell granules and cleaves proteins at arginine and lysine residues. Tryptase is stored and released from mast cell granules upon activation. Mast cells are found in many tissues but are present in greater numbers along epithelial linings of the body, such as the skin, respiratory tract and gastrointestinal tract, as well as the perivascular tissue surrounding blood vessels. They are involved in a variety of physiological and pathophysiological states, including immediate hypersensitivity, delayed-type hypersensitivity, cell growth regulation, defense against neoplasia, and pain and itch sensation. They have also been implicated in chronic inflammatory states and are involved in neuroimmune interactions. The availability of recombinant human tryptase will aid in research directed toward a more complete understanding of the biological role(s) of tryptase and mast cells and the identification of tryptase in vivo targets.
- Harris, J. et al. (2001) J. Biol. Chem. 276, 34941–7.
- Niles, A.L. et al. (1998) Biotechnol. Appl. Biochem. 28, 125–31.