Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana

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.

Activity‐based proteomics reveals nine target proteases for the recombinant protein‐stabilizing inhibitor SlCYS8 in Nicotiana benthamiana

“…Different strategies have been used to suppress unwanted proteolysis in planta (Mandal et al, 2016), but the responsible proteases have not yet been identified. The tomato cystatin SlCYS8 is a stabilizing co-expression partner or a fusion partner for proteins targeted to the plant cell secretory pathway (Grosse-Holz et al, 2018b;Jutras et al, 2016;Sainsbury et al, 2013). In this study, we used SlCYS8 to identify target proteases that may contribute to low yields of foreign recombinant proteins in N. benthamiana.…”

“…Transgenic tobacco plants constitutively expressing a rice cystatin (OC-I) have reduced protease activity, correlated with a higher accumulation of recombinant proteins (Pillay et al, 2012). Transient expression of tomato cystatin SlCYS8 (Girard et al, 2007) in the cell secretory pathway of agroinfiltrated Nicotiana benthamiana leaves also prevents degradation of recombinant proteins, notably leading to higher yields of fully assembled human antibodies (Grosse-Holz et al, 2018b;Jutras et al, 2016;Robert et al, 2013). An inactive version of SlCYS8, which bears a proline (P) instead of a glutamine (Q) in the inhibitory loop QxVxG motif ( Q47P SlCYS8), has no stabilizing effect on recombinant proteins (Grosse-Holz et al, 2018b;Jutras et al, 2016;Sainsbury et al, 2013), indicating that SlCYS8 stabilizes recombinant proteins through inhibiting proteases.…”

Steric Accessibility of the Cleavage Sites Dictates the Proteolytic Vulnerability of the Anti‐HIV‐1 Antibodies 2F5, 2G12, and PG9 in Plants

“…Protein degradation by endogenous proteases complicates recombinant protein production in plant‐based biofactories, which limits the manufacturing of bNAbs and other biotherapeutic proteins in such expression systems . Although proteases occur in virtually all compartments of plant cells, substantial evidence has been presented that the degradation of bNAbs is largely confined to the apoplast .…”

Innovation in plant-based transient protein expression for infectious disease prevention and preparedness

“…Unintended proteolysis is often cited as a concern for plantbased expression and the identification of specific proteolytic activities in the plant secretory pathway allowed rational inhibition of these activities by the co-expression of protease inhibitors (Figure 3a) [32]. This strategy can result in a 1.4-fold increase in recombinant antibody production [33] and consideration of inhibitors regulated in response to plant-pathogen interactions is also uncovering effective protease inhibitors for the protection of recombinant proteins [34 ]. RNAi constructs have also been used to knock down proteolytic activities [35], which has led to a 1.6-fold increase in interleukin 10 accumulation in whole plants [36].…”