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

Abstract

Summary Co‐expression of protease inhibitors like the tomato cystatin Sl CYS 8 is useful to increase recombinant protein production in plants, but key proteases involved in protein proteolysis are still unknown. Here, we performed activity‐based protein profiling to identify proteases that are inhibited by Sl CYS 8 in agroinfiltrated Nicotiana benthamiana . We discovered that Sl CYS 8 selectively suppresses papain‐like cysteine protease ( PLCP ) activity in both apoplastic fluids and total leaf extracts, while not affecting vacuolar‐processing enzyme and serine hydrolase activity. A robust concentration‐dependent inhibition of PLCP s occurred in vitro when purified Sl CYS 8 was added to leaf extracts, indicating direct cystatin– PLCP interactions. Activity‐based proteomics revealed that nine different Cathepsin‐L/‐F‐like PLCP s are strongly inhibited by Sl CYS 8 in leaves. By contrast, the activity of five other Cathepsin‐B/‐H‐like PLCP s, as well as 87 Ser hydrolases, was unaffected by Sl CYS 8. Sl CYS 8 expression prevented protein degradation by inhibiting intermediate and mature isoforms of granulin‐containing proteases from the Resistant‐to‐Desiccation‐21 ( RD 21) P LCP subfamily. Our data underline the key role of endogenous PLCP s on recombinant protein degradation and reveal candidate proteases for depletion strategies.

Homology-guided re-annotation improves the gene models of the alloploidNicotiana benthamiana

“…To confirm that our new annotations are correct and biologically relevant we examined the annotation of 14 papain-like Cys proteases (PLCPs) that we identified recently from agroinfiltrated N. benthamiana leaves (Jutras et al, 2019). These proteases were active proteins because they reacted with an activity-based probe (DCG-04, a biotinylated PLCP inhibitor) to facilitate the purification and detection of these proteins.…”

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

“…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]. The continued identification of specific proteases directly involved in the degradation of recombinant proteins in the secretory pathway increases the potential impact of transient or stable knock-down approaches [37]. An alternative approach to improving protein stability is to modulate the pH of the secretory pathway via the expression of proton channels (Figure 3b).…”

Proteases of Nicotiana benthamiana: an emerging battle for molecular farming

“…The tomato cystatin SlCYS8 was used to improve the yield of fully assembled and biologically active fragments of IgG antibodies transiently expressed in N. benthamiana [19][20][21]. An inactive version of SlCYS8 showed no protective effect on recombinant proteins, indicating that the stabilising effect is accomplished through protease inhibition [20,22]. A chimeric version of SlCYS8, the 'Cysta-tag', has also been designed to combine its inhibition potential with routine protein purification techniques [23].…”

“…Biochemical profiling of active sites using proteome-derived peptide libraries in combination with quantitative proteomics is useful to simultaneously identify N-terminal and C-terminal cleavage motifs [41]. Activity-based protein profiling (ABPP) is also increasingly used to uncover the active proteome using tagged chemical probes that react covalently and irreversibly with the active site of proteins [22,42]. These collective efforts to identify substrates and decipher protease functions will create new opportunities for plant biotechnology applications.…”