Agroinfiltration contributes to VP1 recombinant protein degradation

Abstract

There is a growing interest in applying tobacco agroinfiltration for recombinant protein production in a plant based system. However, in such a system, the action of proteases might compromise recombinant protein production. Protease sensitivity of model recombinant foot-and-mouth disease (FMD) virus P1-polyprotein (P1) and VP1 (viral capsid protein 1) as well as E. coli glutathione reductase (GOR) were investigated. Recombinant VP1 was more severely degraded when treated with the serine protease trypsin than when treated with the cysteine protease papain. Cathepsin L- and B-like as well as legumain proteolytic activities were elevated in agroinfiltrated tobacco tissues and recombinant VP1 was degraded when incubated with such a protease-containing tobacco extract. In silico analysis revealed potential protease cleavage sites within the P1, VP1 and GOR sequences. The interaction modeling of the single VP1 protein with the proteases papain and trypsin showed greater proximity to proteolytic active sites compared to modeling with the entire P1-polyprotein fusion complex. Several plant transcripts with differential expression were detected 24 hr post-agroinfiltration when the RNA-seq technology was applied to identify changed protease transcripts using the recently available tobacco draft genome. Three candidate genes were identified coding for proteases which included the Responsive-to-Desiccation-21 (RD21) gene and genes for coding vacuolar processing enzymes 1a (NbVPE1a) and 1b (NbVPE1b). The data demonstrates that the tested recombinant proteins are sensitive to protease action and agroinfiltration induces the expression of potential proteases that can compromise recombinant protein production.

Plant Serine Protease Inhibitors: Biotechnology Application in Agriculture and Molecular Farming

“…Thus, protein targeting to apoplast may help to correct maturation and glycosylation of recombinant proteins [150]. In addition, several findings suggest that degradation of recombinant proteins expressed in a plant is not related to the extraction process, but along the secretion into the apoplastic space [151]. Therefore, it is important to gain more knowledge of different proteases mainly present in the apoplast in order to develop strategies that would contribute to preventing unintended proteolytic processing and help to the correct the maturation of recombinant proteins [152].…”

“…Although recent studies identified different protease families present in the apoplast space of tomato, A. thaliana , N. benthamiana and N. tabacum [62,153], they require more detailed analyses to confirm their direct involvement in recombinant protein degradation. This information will be useful for planning strategies before recombinant protein production [151]. It will also allow establishing smart protocols combining the introduction of PIs of specific serine, cysteine, aspartic acid, and/or metalloprotease according to the requirements to increase the heterologous protein production.…”

A kiwifruit (Actinidia deliciosa) R2R3‐MYB transcription factor modulates chlorophyll and carotenoid accumulation

“…New Phytologist expression of genes using Agrobacterium infiltration into N. benthamiana as a heterologous host has been used recently to validate gene function or determine protein localisation (Leckie & Stewart, 2011;Pillay et al, 2016). In this study, infiltrated leaves were left for either 24 h (T1) or 72 h (T2) on the plant before tissue was isolated for RNA extraction and analysis by transcriptome sequencing.…”

Identification of two subtilisin‐like serine proteases engaged in the degradation of recombinant proteins in Nicotiana benthamiana

“…However, it is possible that other N. benthamiana SBTs are upregulated upon infection with agrobacteria and thus enhance the proteolytic potential of apoplastic fluid after agroinfiltration. It should be noted that agroinfiltration has been shown to upregulate the expression of papain‐like cysteine proteinases and legumains [33], which have been also implicated in the degradation of recombinant proteins in N. benthamiana and related Nicotiana species [11,20,34].…”