Plant cell suspensions, like Nicotiana tabacum Bright Yellow-2 (BY-2), are established as a platform for producing recombinant glycoproteins such as antibodies and viral glycoproteins. Those glycoproteins are targeted to the secretory pathway and secreted by default into the extracellular medium (EM). However, the presence of some degraded fragments and the removal of protein tags (used for purification) are concerning. We hypothesized that proteases with different catalytic activities act on recombinant glycoproteins as they pass through the cell wall or accumulate in the culture medium.
We identified 59 distinct proteases by mass spectrometry in the EM and the cell wall of BY-2 cells. Most of them are classified as serine or aspartic proteases. Variations in abundance of those proteases in the cell wall and during cell growth were characterized.
To minimize the impact of protease activities on recombinant glycoprotein production, we genetically knocked out their genes using the CRISPR/Cas9 editing system. These knockouts were carried out in a BY-2 cell line secreting two different reporter glycoproteins that are prone to degradation in the EM: a human IgG1 and a viral glycoprotein ectodomain fused to a V5-tag. Two approaches were implemented simultaneously. The first one is based on single gene knockouts and the second one relies on family mutants to prevent the compensatory effect caused by proteases sharing identical activities. The accumulation of intact recombinant glycoproteins and the presence of undamaged V5-tag in mutant cell lines were directly monitored by western blotting. Inactivation of the targeted protease genes was validated by sequencing.