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.

KEYWORDS:

• agroinfiltration
• cysteine proteases
• foot-and-mouth disease
• proteases
• recombinant protein production
• tobacco
• VP1 protein

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Professor Yong Suk Jang, Professor Moon Sik Yang and Dr. Tae Geum Kim for providing us with the VP1 gene and Dr. Huy for assisting with the VP1 purification, Professor George Lomonossoff for providing the P1 construct, and Professor Christine Foyer for providing the GOR construct. We also thank Dr. Francois Maree (Ondersterpoort Veterinary campus, University of Pretoria, South Africa) who kindly provided the Anti-FMDV polyclonal antiserum and Professor Dominique Michaud for assisting us to establish the agroinfiltration technique.

Funding

Our research was supported by the National Research Foundation (NRF) and the Genomics Research Institute (GRI), South Africa as well as NRF incentive funding to Karl Kunert and a NRF bursary to Priyen Pillay.

Notes on contributors

PP and KJK conceptualised and designed the experiment. PP conducted the experiments. BJV financially supported the project and provided analytical tools and scientific intellectual input in data interpretation. MEM helped in running enzymatic assays. CAC and SGVW helped in analyzing the RNAseq data and manuscript reading. All authors read and approved the manuscript.