Companion Protease Inhibitors for the In Situ Protection of Recombinant Proteins in Plants

Abstract

We previously described a procedure for the use of plant protease inhibitors as "companion" accessory proteins to prevent unwanted proteolysis of clinically useful recombinant proteins in leaf crude protein extracts (Benchabane et al. Methods Mol Biol 483:265-273, 2009). Here we describe the use of these inhibitors for the protection of recombinant proteins in planta, before their extraction from leaf tissues. A procedure is first described involving inhibitors co-expressed along-and co-migrating-with the protein of interest in host plant cells. An alternative, single transgene scheme is then described involving translational fusions of the recombinant protein and companion inhibitor. These approaches may allow for a significant improvement of protein steady-state levels in leaves, comparable to yield improvements observed with protease-deficient strains of less complex protein expression hosts such as E. coli or yeasts.

An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves

“…As previously observed for a number of heterologous proteins co-expressed with other proteins [30], α 1 -ACT had a negative impact on yields of both the heavy and light chains (A lanes on Fig 2B). By comparison, Sl CYS8 had little impact on light chain accumulation but a strong positive impact on heavy chain-containing assemblies, especially in upper and middle leaves (S lanes on Fig 2B).…”

“…benthamiana leaves. They also suggested a negative impact of protease inhibitor co-expression on IgG accumulation as observed with other proteins [30,36], strongly compensated by the heavy chain-stabilizing effect of co-secreted Sl CYS8 in upper and middle leaves.…”

“…One approach along this line consists of silencing host protease forms using DNA antisense or RNAi sequences [26–28]. Another approach consists of co-expressing accessory protease inhibitors with the protein of interest to inhibit endogenous protease activities in situ [29, 30]. Co-secretion of tomato cystatins Sl CYS8 or Sl CYS9, two Cys protease inhibitors, was shown for instance to improve the accumulation of a transiently expressed diagnostic monoclonal IgG in N .…”

Recombinant protein susceptibility to proteolysis in the plant cell secretory pathway is pH‐dependent

“…We here report a complementary effect of the viral channel, by which proton transport out of the Golgi influences the integrity of acid-tolerant recombinant proteins via an indirect, pH-related effect on resident protease activities. Work is underway to detect eventual synergistic effects between this new way of modulating endogenous protease activity patterns and previously described strategies for the ectopic inhibition of specific protease family targets Robert et al, 2016). Research efforts will be welcome in coming years to evaluate the feasibility of combining the mCystaTag affinity handle, M2 channel co-expression and protease-stable linkers such as Linker F or Linker C to improve the yield of recombinant proteins in planta and facilitate their purification following extraction from leaf tissue.…”

“…A fusion protein system for peptide cleavage monitoring in the cell secretory pathway Several studies have described the negative effects of host resident proteases on the stability of secreted proteins in plant expression systems (Benchabane et al, 2008;Doran, 2006). Several studies have also described strategies to alleviate this problem, notably involving the silenced expression of endogenous protease targets (Duwadi et al, 2015;Mandal et al, 2014) or the co-expression of accessory protease inhibitors to prevent unintended proteolysis in situ Robert et al, 2016). We here assessed whether the post-ER-positive effect of M2 channel on secreted protein (e.g.…”