Extracellular peptidase hunting for improvement of protein production in plant cells and roots

Plant-based biopharmaceuticals have gained a lot of interest in the past decade due to their reduced cost and relative safety compared to mammalian cell cultures. While the first plant-made recombinant proteins are now reaching the market, the production systems still need improvements to maximize their competitiveness, proteolysis being one of the main factors limiting the yields. Identifying and inhibiting in vivo endogenous proteases involved in the degradation of recombinant proteins could then lead to a significant increase in production yields.
In this study, we focused on two different production systems in Arabidopsis thaliana: rhizosecretion and cell suspensions. Extracellular proteases of both systems were used in vitro to study the conditions of target protein degradation (Bovine Serum Albumine, BSA). First, proteases from both systems degrade BSA at both acidic and neutral-to-basic pH conditions. Then, serine and metallopeptidases were shown to be the main protease classes responsible for BSA degradation by rhizosecreted proteomes or extracellular cell culture media, respectively. Finally, the biochemical tests were coupled to a bioinformatics analysis of publicly available transcriptomic data, in order to reduce the number of the proteases most likely involved in BSA degradation. Using this method, only five serine proteases and two metallopeptidases remain candidates for an amiRNA-mediated in vivo inhibition.