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 modelling of the single VP1 protein with the proteases papain and trypsin showed greater
proximity to proteolytic active sites compared to modelling 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.
