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Secreted expression of pseudozymogen forms of recombinant matriptase in Pichia pastoris

Paper ID Volume ID Publish Year Pages File Format Full-Text
17689 42689 2009 7 PDF Available
Title
Secreted expression of pseudozymogen forms of recombinant matriptase in Pichia pastoris
Abstract

Matriptase is a transmembrane serine protease expressed in vertebrates. This enzyme is synthesized as a zymogen form and is converted to an active form by cleavage at the N-terminus of the serine protease catalytic domain. In a mammalian cell-based expression system, we have produced pseudozymogen forms of recombinant matriptase (r-matriptase) that are activated by cleavage with a recombinant enterokinase (r-EK) in vitro. In the present study, four different pseudozymogen forms of r-matriptase containing a site for activation by r-EK and a hexahistidine tag (His6-tag) were expressed in and secreted by Pichia pastoris, a methylotrophic yeast. The pseudozymogens with His6-tag at their C-termini formed multimers linked by intermolecular disulfide bonds. After treatment with r-EK, they exhibited no detectable hydrolytic activity toward a chromogenic substrate. A pseudozymogen form of matriptase catalytic domain with N-terminal His6-tag (designated His6t-S-CD) was secreted as a monomer. His6t-S-CD after r-EK treatment exhibited activity comparable to that of the activated form of an r-matriptase expressed in mammalian cells. His6t-S-CD could be purified from culture medium in milligram quantities. The expression in the yeast offers an efficient method of producing larger amounts of r-matriptase.

Keywords
Catalytic domain; Matriptase; Pichia pastoris; Type II transmembrane serine protease
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Secreted expression of pseudozymogen forms of recombinant matriptase in Pichia pastoris
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 45, Issue 4, 7 October 2009, Pages 288–294
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us