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GreenProteases Report Summary

Project ID: 616449
Funded under: FP7-IDEAS-ERC
Country: United Kingdom

Periodic Report Summary 2 - GREENPROTEASES (The Proteolytic Machinery of the Plant Apoplast:from Basic Understanding to Improved Recombinant Protein Production)

Agroinfiltration of Nicotiana benthamiana is frequently used in Plant Science to study plant proteins and in Molecular Pharming to produce recombinant proteins. However, this protein expression platform is restricted by endogenous proteases, which limit the accumulation levels of most proteins. We aim to understand the proteolytic machinery of agroinfiltrated N. benthamiana plants, with a focus on the extracellular space, the apoplast. Using RNAseq we found that agroinfiltrated leaves express 1044 putative proteases. Proteomics of extracellular proteomes showed that the apoplast contains at least 196 putative proteases, and protease activity profiling showed that 54 of these to be active. Collectively, these proteases destabilise a broad range of our tested recombinant proteins including antibodies glycohormones and enzymes. Protease depletion by RNAi approaches have did not increase recombinant protein expression, presumably because of the redundancy in the proteolytic machinery. Indeed, even cocktails of broad-range chemical protease inhibitors could not stop recombinant protein degradation. By contrast, protease depletion by co-expressing protein-based inhibitors increases protein accumulation, and various approaches are undertaken to identify the proteases targeted by these inhibitors. We are also using CRISPR/Cas9 to generate protease-depleted lines to identify natural substrates and to combine with other protease depletion methods to untangle the complex and redundant proteolytic machinery of agroinfiltrated N. benthamiana.

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United Kingdom
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