Production of Desmodus rotundus salivary plasminogen activator α1 (DSPAα1) in tobacco is hampered by proteolysis

Authors

  • Andreas Schiermeyer,

    1. Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie (IME), c/o Institut für Biologie VII, RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany; telephone: 49-241-80-28128; fax: 49-241-871062
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  • Helga Schinkel,

    1. Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie (IME), c/o Institut für Biologie VII, RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany; telephone: 49-241-80-28128; fax: 49-241-871062
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  • Stefanie Apel,

    1. Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie (IME), c/o Institut für Biologie VII, RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany; telephone: 49-241-80-28128; fax: 49-241-871062
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  • Rainer Fischer,

    1. Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie (IME), c/o Institut für Biologie VII, RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany; telephone: 49-241-80-28128; fax: 49-241-871062
    2. Rheinisch-Westfälische Technische Hochschule (RWTH), Institut für Biologie VII (Molekulare Biotechnologie), Worringerweg 1, 52074 Aachen, Germany
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  • Stefan Schillberg

    Corresponding author
    1. Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie (IME), c/o Institut für Biologie VII, RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany; telephone: 49-241-80-28128; fax: 49-241-871062
    • Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie (IME), c/o Institut für Biologie VII, RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany; telephone: 49-241-80-28128; fax: 49-241-871062
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  • Abbreviations: CHO, Chinese hamster ovary; DSPA, Desmodus rotundus salivary plasminogen activator; IMAC, immobilized metal affinity chromatography; mAb, monoclonal antibody; NBT/BCIP, nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate; pNA, para-nitroanilin

Abstract

The high fibrin specificity of Desmodus rotundus salivary plasminogen activator α1 (DSPAα1 or desmoteplase (INN)) makes it a promising candidate for the treatment of acute ischemic stroke. In the current study we explored the use of transgenic tobacco plants and BY-2 suspension cells as alternative production platforms for this drug. Four different N-terminal signal peptides, from plants and animals, were used to translocate the recombinant DSPAα1 protein to the endomembrane system. Intact recombinant DSPAα1 was produced in transgenic plants and BY-2 cells, although a certain degree of degradation was observed in immunoblotted extracts. The choice of signal peptide had no major influence on the degradation pattern or recombinant protein accumulation, which reached a maximum level of 38 μg/g leaf material. N-terminal sequencing of purified, His6-tagged DSPAα1 revealed only minor changes in the position of signal peptide cleavage compared to the same protein expressed in Chinese hamster ovary cells. However, correctly processed recombinant DSPAα1 was also detected. The enzymatic activity of the recombinant protein was confirmed using an in vitro assay with unpurified and purified samples, demonstrating that plants are suitable for the production of functional DSPAα1. In contrast to whole plant cell extracts, no recombinant DSPAα1 was detected in the culture supernatant of transgenic BY-2 cells. Further analysis showed that recombinant DSPAα1 is subject to proteolysis and that endogenous secreted BY-2 proteases are responsible for DSPAα1 degradation in the culture medium. The addition of a highly concentrated protease inhibitor mixture or 5 mM EDTA reduced DSPAα1 proteolysis, improving the accumulation of intact product in the culture medium. Strategies to improve the plant cell suspension system for the production of secreted recombinant proteins are discussed. © 2005 Wiley Periodicals, Inc.

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