title: Production of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAalpha1) in tobacco is hampered by proteolysis.
abstract: The high fibrin specificity of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAalpha1 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 DSPAalpha1 protein to the endomembrane system. Intact recombinant DSPAalpha1 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 microg/g leaf material. N-terminal sequencing of purified, His6-tagged DSPAalpha1 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 DSPAalpha1 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 DSPAalpha1. In contrast to whole plant cell extracts, no recombinant DSPAalpha1 was detected in the culture supernatant of transgenic BY-2 cells. Further analysis showed that recombinant DSPAalpha1 is subject to proteolysis and that endogenous secreted BY-2 proteases are responsible for DSPAalpha1 degradation in the culture medium. The addition of a highly concentrated protease inhibitor mixture or 5 mM EDTA reduced DSPAalpha1 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.
authors.affiliation: Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie (IME), c/o Institut für Biologie VII, RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schiermeyer
authors.forename: Helga
authors.initials: H
authors.lastname: Schinkel
authors.forename: Stefanie
authors.initials: S
authors.lastname: Apel
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.forename: Stefan
authors.initials: S
authors.lastname: Schillberg
mesh_terms: D015153:Blotting, Western; D005240:Feasibility Studies; D006639:Histidine / Q000737:chemistry; D006868:Hydrolysis; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D010960:Plasminogen Activators / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; C069588:salivary plasminogen activator alpha 1, Desmodus rotundus; D006639:Histidine; D010960:Plasminogen Activators