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.
Highlights
Plant-based protein expression platforms are a useful answer to the growing demand for biological therapeutics and diagnostics worldwide [1,2]
N. benthamiana as an expression host was first characterised in terms of leaf biomass for protein extraction, in order to gain insight into the relationship between physiological state along the leaf-age gradient and recombinant protein yield
In the current context of therapeutic protein production where both yield and consistent quality are important for the development of plant-based production platforms [1,44], there is a need to elucidate metabolic processes governing the production and modification of recombinant proteins in planta and to understand how these processes are affected by plant development and physiology
Summary
Plant-based protein expression platforms are a useful answer to the growing demand for biological therapeutics and diagnostics worldwide [1,2]. Most useful as a method to quickly screen transgene expression constructs [7], transient expression in agroinfiltrated plant leaves has developed into the fastest and most convenient production platform for plant-made biopharmaceuticals [8,9] This technique takes advantage of the ability of Agrobacterium species to transfer a transcriptionally competent segment of DNA, the T-DNA, into the plant host cell, where it is directed to the nucleus [10]. Well known as a model for studies on plant-pathogen interactions [16], N. benthamiana grows quickly and is amenable to agroinfiltration for high-level protein production As a result, this species has become somewhat of a model plant for the transient expression of foreign proteins
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