Abstract
SummaryAgroinfiltrated Nicotiana benthamiana is a flexible and scalable platform for recombinant protein (RP) production, but its great potential is hampered by plant proteases that degrade RPs. Here, we tested 29 candidate protease inhibitors (PIs) in agroinfiltrated N. benthamiana leaves for enhancing accumulation of three unrelated RPs: glycoenzyme α‐Galactosidase; glycohormone erythropoietin (EPO); and IgG antibody VRC01. Of the previously described PIs enhancing RP accumulation, we found only cystatin SlCYS8 to be effective. We identified three additional new, unrelated PIs that enhance RP accumulation: N. benthamiana NbPR4, NbPot1 and human HsTIMP, which have been reported to inhibit cysteine, serine and metalloproteases, respectively. Remarkably, accumulation of all three RPs is enhanced by each PI similarly, suggesting that the mechanism of degradation of unrelated RPs follows a common pathway. Inhibitory functions HsTIMP and SlCYS8 are required to enhance RP accumulation, suggesting that their target proteases may degrade RPs. Different PIs additively enhance RP accumulation, but the effect of each PI is dose‐dependent. Activity‐based protein profiling (ABPP) revealed that the activities of papain‐like Cys proteases (PLCPs), Ser hydrolases (SHs) or vacuolar processing enzymes (VPEs) in leaves are unaffected upon expression of the new PIs, whereas SlCYS8 expression specifically suppresses PLCP activity only. Quantitative proteomics indicates that the three new PIs affect agroinfiltrated tissues similarly and that they all increase immune responses. NbPR4, NbPot1 and HsTIMP can be used to study plant proteases and improve RP accumulation in molecular farming.
Highlights
Molecular farming, the production of biopharmaceuticals in plants, offers speed, scalability and low risk of contamination with human pathogens when compared to insect or mammalian cell culture systems (Stoger et al, 2014)
To overcome the degradation bottleneck in molecular farming, we aimed to co-express secreted recombinant proteins (RPs) with secreted protease inhibitors (PIs)
We identified four endogenous PIs associated with plant immunity, reasoning that they might both inhibit proteases and enhance plant fitness during interaction with A. tumefaciens
Summary
The production of biopharmaceuticals in plants, offers speed, scalability and low risk of contamination with human pathogens when compared to insect or mammalian cell culture systems (Stoger et al, 2014). N. benthamiana leaves can be genetically modified by infiltration with disarmed Agrobacterium tumefaciens (Agrobacterium) carrying gene(s) of interest on the transfer DNA (T-DNA) of binary plasmid(s) (Bevan, 1984). Agrobacterium delivers the T-DNA to the plant nucleus, allowing foreign genes to be transiently expressed. Co-expression of several transgenes is achieved by mixing Agrobacterium cultures delivering different transgenes before agroinfiltration. Agroinfiltration-mediated protein expression can deliver ten million doses of the latest influenza vaccine within 6 weeks (Pillet et al, 2016). To maximize efficacy and limit immunogenicity, biopharmaceuticals like ZMapp are produced with humanized N-glycans in the secretory pathway of genetically engineered N. benthamiana (Castilho et al, 2014; Schoberer and Strasser, 2017)
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