Protection of Recombinant Mammalian Antibodies from Development-Dependent Proteolysis in Leaves of Nicotiana benthamiana

Abstract

The expression of clinically useful proteins in plants has been bolstered by the development of high-yielding systems for transient protein expression using agroinfiltration. There is a need now to know more about how host plant development and metabolism influence the quantity and quality of recombinant proteins. Endogenous proteolysis is a key determinant of the stability and yield of recombinant proteins in plants. Here we characterised cysteine (C1A) and aspartate (A1) protease profiles in leaves of the widely used expression host Nicotiana benthamiana, in relation with the production of a murine IgG, C5-1, targeted to the cell secretory pathway. Agroinfiltration significantly altered the distribution of C1A and A1 proteases along the leaf age gradient, with a correlation between leaf age and the level of proteolysis in whole-cell and apoplast protein extracts. The co-expression of tomato cystatin SlCYS8, an inhibitor of C1A proteases, alongside C5-1 increased antibody yield by nearly 40% after the usual 6-days incubation period, up to ∼3 mg per plant. No positive effect of SlCYS8 was observed in oldest leaves, in line with an increased level of C1A protease activity and a very low expression rate of the inhibitor. By contrast, C5-1 yield was greater by an additional 40% following 8- to 10-days incubations in younger leaves, where high SlCYS8 expression was maintained. These findings confirm that the co-expression of recombinant protease inhibitors is a promising strategy for increasing recombinant protein yields in plants, but that further opportunity exists to improve this approach by addressing the influence of leaf age and proteases of other classes.

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

“…N . benthamiana plants with eight leaves arranged in an alternate pattern along the main stem were previously proposed to include three morphological zones encompassing, respectively, young growing leaves (leaves 1, 2 and 3), mature leaves (leaves 4, 5 and 6) and older–including senescent–leaves (leaves 7 and 8) [31]. We numbered the leaves using the same pattern, from the apex down, with the 'Upper' zone corresponding to young leaves, the 'Middle' zone to mature leaves and the 'Bottom' zone to older leaves (Fig 2A).…”

“…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 . benthamiana leaves [13, 31]. Similarly, a soybean Ser protease inhibitor secreted by the hairy roots of transgenic tobacco lines was shown to stabilize the light and heavy chains of IgG variants co-secreted in the hydroponic culture medium [32].…”

Assembly and Purification of Polyomavirus-Like Particles from Plants

“…Proteolysis can often be a limiting factor for expression in plant cells [5], and it may be possible to address this issue through a number of strategies. For example, the stable or transient co-expression of serine protease inhibitors as has been demonstrated for aspartic proteases in the cytosol [16] and cysteine proteases in the secretory pathway [33], or by post-transcriptionally regulating endogenous protease levels as recently demonstrated with anti-sense RNA inhibition [22].…”

“…To minimise the variability of protein expression levels from leaves of different ages [33], the experiments were conducted to allow sampling from at least three different leaves per construct. Infiltrated leaf tissue was harvested as leaf discs equivalent to 120 mg of control-infiltrated tissue, and homogenised by disrupting tissue with 7-mm tungsten beads using a TissueLyserII (Qiagen, www.qiagen.com) with prechilled tube holders in three volumes of phosphate-buffered saline (PBS), pH 7.3, containing 5 mM ethylenediaminetetra-acetic acid (EDTA), 0.05 % (v/v) Triton X-100, and complete protease inhibitor cocktail (Roche, www.roche.…”

Molecular farming on rescue of pharma industry for next generations

“…This hurdle, however, can be circumvented by rapid processing of leaves immediately upon harvest and by protecting recombinant proteins from development-dependent proteolysis in leaves (Robert et al, 2013). Other important leafy crops include: lettuce, alfalfa and soybean.…”