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Identification, gene cloning and expression of serine proteases in the extracellular medium of Nicotiana tabacum cells

Plant Cell Reports volume 31, pages 1959–1968 (2012)Cite this article

Abstract

Recombinant proteins secreted from plant suspension cells into the medium are susceptible to degradation by host proteases secreted during growth. Some degradation phenomena are inhibited in the presence of various protease inhibitors, such as EDTA or AEBSF/PMSF, suggesting the presence of different classes of proteases in the medium. Here, we report the results of a proteomic analysis of the extracellular medium of a Nicotiana tabacum bright yellow 2 culture. Several serine proteases belonging to a Solanaceae-specific subtilase subfamily were identified and the genes for four cloned. Their expression at the RNA level during culture growth varied depending on the gene. An in-gel protease assay (zymography) demonstrated serine protease activity in the extracellular medium from cultures. This was confirmed by testing the degradation of an antibody added to the culture medium. This particular subtilase subfamily, therefore, represents an interesting target for gene silencing to improve recombinant protein production.

Key message The extracellular medium of Nicotiana tabacum suspension cells contains serine proteases that degrade antibodies.

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Acknowledgments

This work was supported in part by grants from the Service public de Wallonie (Waleo3 08/1/6861) and the European community (PharmaPlanta integrated project). BM is the recipient of a fellowship from the Fonds pour la Formation à la Recherche dans l’Industrie et dans l’Agriculture (Belgium). DV is a Collaborateur Logistique of the FRS-FNRS (Belgium).

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Affiliations

  1. Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud 4–5, 1348, Louvain la Neuve, Belgium

    Catherine Navarre, Benoit De Muynck, Georges Alves, Bertrand Magy & Marc Boutry

  2. Institut de Duve, Université catholique de Louvain, Avenue Hippocrate 75, 1200, Woluwé Saint Lambert, Belgium

    Didier Vertommen

Authors
  1. Catherine Navarre
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  2. Benoit De Muynck
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  3. Georges Alves
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  4. Didier Vertommen
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  5. Bertrand Magy
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  6. Marc Boutry
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Corresponding author

Correspondence to Marc Boutry.

Additional information

Communicated by S. Schillberg.

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Navarre, C., De Muynck, B., Alves, G. et al. Identification, gene cloning and expression of serine proteases in the extracellular medium of Nicotiana tabacum cells. Plant Cell Rep 31, 1959–1968 (2012). https://doi.org/10.1007/s00299-012-1308-y

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  • DOI: https://doi.org/10.1007/s00299-012-1308-y

Keywords

  • Cell suspension culture
  • BY-2 cells
  • Serine proteases
  • Nicotiana tabacum