Activity‐based proteomics reveals nine target proteases for the recombinant protein‐stabilizing inhibitor Sl CYS 8 in Nicotiana benthamiana

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.