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Production of Functional Plant Legumain Proteases Using the Leishmania tarentolae Expression System

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Plant Proteases and Plant Cell Death

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2447))

Abstract

Plant proteases of the legumain-type are key players in many processes along the plant life cycle. In particular, legumains are especially important in plant programmed cell death and the processing and maturation of seed storage proteins within the vacuole. Plant legumains are therefore synonymously called vacuolar processing enzymes (VPEs). Because of their dual protease and cyclase activities, plant legumains are of great interest to biotechnological applications, e.g., for the development of cyclic peptides for drug design. Despite this high interest by the scientific community, the recombinant expression of plant legumains proved challenging due to several posttranslational modifications, including (1) the formation of structurally critical disulfide bonds, (2) activation via pH-dependent proteolytic processing, and (3) stabilization by varying degrees of glycosylation. Recently we could show that LEXSY is a robust expression system for the production of plant legumains. Here we provide a general protocol for the recombinant expression of plant legumains in Leishmania cells. We further included detailed procedures for legumain purification, activation and subsequent activity assays and additionally note specific considerations with regard to isoform specific activation intermediates. This protocol serves as a universal strategy for different legumain isoforms from different source organisms.

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Acknowledgments

This work was supported by funding from the Austrian Science Fund (FWF, project numbers W_01213 and P31867).

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Authors and Affiliations

  1. Department of Biosciences, University of Salzburg, Salzburg, Austria

    Elfriede Dall & Hans Brandstetter

  2. Jena Bioscience, Jena, Germany

    Andreas Licht

Authors
  1. Elfriede Dall
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  2. Andreas Licht
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  3. Hans Brandstetter
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Corresponding author

Correspondence to Elfriede Dall .

Editor information

Editors and Affiliations

  1. Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia

    Marina Klemenčič

  2. VIB-UGent Cntr for Plant Systems Biology, Ghent University, Ghent, Belgium

    Simon Stael

  3. ZEA-3 Analytik, Forschungszentrum Juelich, Juelich, Germany

    Pitter F. Huesgen

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Dall, E., Licht, A., Brandstetter, H. (2022). Production of Functional Plant Legumain Proteases Using the Leishmania tarentolae Expression System. In: Klemenčič, M., Stael, S., Huesgen, P.F. (eds) Plant Proteases and Plant Cell Death. Methods in Molecular Biology, vol 2447. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2079-3_4

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  • DOI: https://doi.org/10.1007/978-1-0716-2079-3_4

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2078-6

  • Online ISBN: 978-1-0716-2079-3

  • eBook Packages: Springer Protocols

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