Protein-Derived Signal Peptides Induced by Agrobacterium Infection Promote the Secretion of Recombinant Proteins in Nicotiana benthamiana

Abstract

Plants are promising next-generation hosts for recombinant protein production; however, major challenges remain with regard to enhancing the efficiency of downstream processing, particularly in the removal of cellular residues and purification of the expressed proteins. Strategies to overcome these limitations include targeting expressed recombinant proteins within a specific organelle or directing their secretion into the extracellular space, thereby facilitating purification by collecting the target matrix. In this study, we focused on protein secretion mechanisms and identified two pathogenesis-related proteins, glucan endo-1,3-β-glucosidase (GN) and chitinase 8 (Chi8), which accumulated in the apoplast washing fluid following Agrobacterium infiltration of Nicotiana benthamiana leaves. Both proteins contained signal peptides (SPs), SP<inf>GN</inf> and SP<inf>Chi8</inf>, respectively. Although the intracellular accumulation of GFP was comparable regardless of the expression level, fusion with either SP<inf>GN</inf> or SP<inf>Chi8</inf> resulted in GFP accumulation within the apoplast. In contrast, in N. benthamiana, a mammalian-derived SP was less effective in facilitating GFP secretion than the plant-derived SPs. Additionally, replacing the SP of the mammalian-derived protein β-glucocerebrosidase (GCase) with SP<inf>GN</inf> or SP<inf>Chi8</inf> enhanced the secretion of GCase into the apoplast, indicating their applicability in protein production. Moreover, SP<inf>GN</inf> and SP<inf>Chi8</inf> directed the expressed proteins into the culture medium of N. benthamiana suspension cells. These results indicate that SP<inf>GN</inf> and SP<inf>Chi8</inf> function as effective secretion signals and highlight the potential application of endogenous SPs for enhancing recombinant protein production in plants.