Open AccessThree unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana
Author(s) -
GrosseHolz Friederike,
Madeira Luisa,
Zahid Muhammad Awais,
Songer Molly,
Kourelis Jiorgos,
Fesenko Mary,
Ninck Sabrina,
Kaschani Farnusch,
Kaiser Markus,
Hoorn Renier A.L.
Publication year - 2018
Publication title -
plant biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.525
H-Index - 115
eISSN - 1467-7652
pISSN - 1467-7644
DOI - 10.1111/pbi.12916
Subject(s) - nicotiana benthamiana , proteases , biology , cystatin , protease , recombinant dna , serine protease , cysteine protease , biochemistry , serine , enzyme , microbiology and biotechnology , cystatin c , gene , renal function
Summary Agroinfiltrated Nicotiana benthamiana is a flexible and scalable platform for recombinant protein ( RP ) production, but its great potential is hampered by plant proteases that degrade RP s. Here, we tested 29 candidate protease inhibitors ( PI s) in agroinfiltrated N. benthamiana leaves for enhancing accumulation of three unrelated RP s: glycoenzyme α‐Galactosidase; glycohormone erythropoietin ( EPO ); and IgG antibody VRC 01. Of the previously described PI s enhancing RP accumulation, we found only cystatin Sl CYS 8 to be effective. We identified three additional new, unrelated PI s that enhance RP accumulation: N. benthamiana Nb PR 4, NbPot1 and human Hs TIMP , which have been reported to inhibit cysteine, serine and metalloproteases, respectively. Remarkably, accumulation of all three RP s is enhanced by each PI similarly, suggesting that the mechanism of degradation of unrelated RP s follows a common pathway. Inhibitory functions Hs TIMP and Sl CYS 8 are required to enhance RP accumulation, suggesting that their target proteases may degrade RP s. Different PI s 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 ( PLCP s), Ser hydrolases ( SH s) or vacuolar processing enzymes ( VPE s) in leaves are unaffected upon expression of the new PI s, whereas Sl CYS 8 expression specifically suppresses PLCP activity only. Quantitative proteomics indicates that the three new PI s affect agroinfiltrated tissues similarly and that they all increase immune responses. Nb PR 4, NbPot1 and Hs TIMP can be used to study plant proteases and improve RP accumulation in molecular farming.