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Inactivation of extracellular peptidase genes as a promising strategy to improve recombinant glycoprotein production in tobacco BY-2 cell cultures by Mathilde MOENS -
Vendredi 13 février 2026 à 16h30 - Salle Jean-Baptiste Carnoy (B.059) - Bâtiment Carnoy - Croix du Sud - 1348 Louvain-la-neuve -
Plant cell suspensions, such as Nicotiana tabacum BY-2 cells, represent a promising platform for the production of recombinant glycoproteins. However, accumulation of intact recombinant protein is impaired by proteolytic degradation processes in culture media. The identification of extracellular proteases involved in these degradation pathways are therefore essential for improving protein yields in plant-based systems.
In this work, peptidase gene inactivation using CRISPR/Cas9 editing was investigated as a strategy to prevent the degradation of recombinant proteins by extracellular peptidases. This strategy was applied to a BY-2 cell line secreting two degradation-prone glycoproteins of pharmaceutical interest: a human IgG1 and a glycoprotein ectodomain from a virus (VGE).
First, extracellular peptidases were identified in samples from the culture medium and the cell wall. Cell wall samples were obtained using an optimized, CaCl₂-based cell wall protein extraction protocol. We identified 53 extracellular peptidases using mass spectrometry.
Based on these results, we attempted to knock out sixteen subtilase genes in TiO5 cells, of which fourteen were successfully inactivated. We also inactivated two out of four metallopeptidase genes in wild-type BY-2 cells. Accumulation of IgG1 and VGE was improved in subtilase-depleted cell lines. Additionally, we compared the proteolytic activity of wild-type, TiO5, and peptidase knockout cell lines and demonstrated that peptidase gene inactivation led to a significant reduction in proteolytic activity in the culture medium.
Collectively, these results demonstrate that peptidase engineering in plant cells represents a promising strategy to alleviate recombinant protein proteolytic degradation, although it requires further investigation. This will enable the further development of BY-2 cells as a robust platform for RP production.
Jury members :
Prof. François CHAUMONT (UCLouvain) (Supervisor)
Dr. Catherine NAVARRE (UCLouvain) (Co-supervisor)
Prof. Michel GHISLAIN (UCLouvain) (Chairperson)
Prof. Henri BATOKO (UCLouvain) (Secretary)
Prof. Laurent GATTO (UCLouvain)
Prof. Frank VAN BREUSEGEM (UGent)
Prof. Muriel BARDOR (Univ. Rouen, Fr.)
Pay attention : the public defense of Mathilde MOENS will also take place in the form of a videoconference
