Enabling Low Cost Biopharmaceuticals: A Systematic Approach to Delete Proteases from a Well-Known Protein Production Host Trichoderma reesei

Christopher P. Landowski*, Anne Huuskonen, Ramon Wahl, Ann Westerholm-Parvinen, Anne Kanerva, Anna-Liisa Hanninen, Noora Salovuori, Merja Penttilä, Jari Natunen, Christian Ostermeier, Bernhard Helk, Juhani Saarinen, Markku Saloheimo

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

Abstract

The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefore, it would be an excellent host for producing high levels of therapeutic proteins at low cost. Developing a filamentous fungus to produce sensitive therapeutic proteins requires that protease secretion is drastically reduced. We have identified 13 major secreted proteases that are related to degradation of therapeutic antibodies, interferon alpha 2b, and insulin like growth factor. The major proteases observed were aspartic, glutamic, subtilisin-like, and trypsin-like proteases. The seven most problematic proteases were sequentially removed from a strain to develop it for producing therapeutic proteins. After this the protease activity in the supernatant was dramatically reduced down to 4% of the original level based upon a casein substrate. When antibody was incubated in the six protease deletion strain supernatant, the heavy chain remained fully intact and no degradation products were observed. Interferon alpha 2b and insulin like growth factor were less stable in the same supernatant, but full length proteins remained when incubated overnight, in contrast to the original strain. As additional benefits, the multiple protease deletions have led to faster strain growth and higher levels of total protein in the culture supernatant.

Original languageEnglish
Article number0134723
Number of pages28
JournalPloS one
Volume10
Issue number8
DOIs
Publication statusPublished - 26 Aug 2015
MoE publication typeA1 Journal article-refereed

Keywords

  • FACTORS INFLUENCING GLYCOSYLATION
  • ASPERGILLUS-NIGER
  • EXTRACELLULAR PROTEASES
  • TRANSFORMATION SYSTEM
  • HYPOCREA-JECORINA
  • HETEROLOGOUS PROTEINS
  • SERINE-PROTEASE
  • N-GLYCOSYLATION
  • O-GLYCOSYLATION
  • HUMAN LYSOZYME

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