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

## TL;DR

Plant proteins called signal peptides, when used, help in secreting desired proteins more efficiently in plants, making protein production easier.

## Contribution

Identified plant-derived signal peptides that enhance recombinant protein secretion in Nicotiana benthamiana.

## Key findings

- SPGN and SPChi8 direct proteins to the apoplast in Nicotiana benthamiana.
- Replacing mammalian signal peptides with SPGN or SPChi8 improves recombinant protein secretion.
- Plant-derived signal peptides are more effective than mammalian ones in this system.

## 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), SPGN and SPChi8, respectively. Although the intracellular accumulation of GFP was comparable regardless of the expression level, fusion with either SPGN or SPChi8 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 SPGN or SPChi8 enhanced the secretion of GCase into the apoplast, indicating their applicability in protein production. Moreover, SPGN and SPChi8 directed the expressed proteins into the culture medium of N. benthamiana suspension cells. These results indicate that SPGN and SPChi8 function as effective secretion signals and highlight the potential application of endogenous SPs for enhancing recombinant protein production in plants.