Abstract

The recovery of recombinant proteins from plant tissues is an expensive and time-consuming process involving plant harvesting, tissue extraction, and subsequent protein purification. The downstream process costs can represent up to 80% of the total cost of production. Secretion-based systems of carnivorous plants might help circumvent this problem. Drosera and Nepenthes can produce and excrete out of their tissues a digestive fluid containing up to 200 mg. L-1 of natural proteins. Based on the properties of these natural bioreactors, we have evaluated the possibility to use carnivorous plants for the production of recombinant proteins. In this context, we have set up original protocols of stable and transient genetic transformation for both Drosera and Nepenthes sp. The two major drawbacks concerning the proteases naturally present in the secretions and a polysaccharidic network composing the Drosera glue were overcome by modulating the pH of the plant secretions. At alkaline pH, digestive enzymes are inactive and the interactions between the polysaccharidic network and proteins in the case of Drosera are subdued allowing the release of the recombinant proteins. For D. capensis, a concentration of 25 μg of GFP/ml of secretion (2% of the total soluble proteins from the glue) was obtained for stable transformants. For N. alata, a concentration of 0.5 ng of GFP/ml secretions (0.5% of total soluble proteins from secretions) was reached, corresponding to 12 ng in one pitcher after 14 days for transiently transformed plants. This plant-based expression system shows the potentiality of biomimetic approaches leading to an original production of recombinant proteins, although the yields obtained here were low and did not allow to qualify these plants for an industrial platform project.

Highlights

  • Development of efficient and cost-competitive expression systems for the production of recombinant proteins constitutes a scientific and an important economical challenge in the field of biotechnologies

  • Molecular Characterization of Transgenic D. capensis Establishment of an Agrobacterium-mediated transformation protocol and subsequent regeneration of transgenic plants require setting up several parameters such as an optimal hormonal balance adapted to plant regeneration, the selection of transformed cells and parameters linked to transfection of T-DNA from agrobacteria to plant cells

  • To regenerate D. capensis plantlets from leaf explants we only used low quantity of BAP (0.005 mg/L) in contrary to D. rotundifolia that needed a combination of auxin and cytokinin (0.45 mg/L BAP and 0.372 mg/L 1-Naphthaleneacetic acid)

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Summary

Introduction

Development of efficient and cost-competitive expression systems for the production of recombinant proteins constitutes a scientific and an important economical challenge in the field of biotechnologies. Elelyso produced in disposable bioreactors by an engineered carrot plant root cell line (ProCellEx R ) and used for the treatment of Gaucher’s disease, was the first recombinant glycoprotein produced by a plant system and has been commercialized since 2012 (Mizukami et al, 2018). This plant cell system has been used to produce Pegunigalsidase α (treatment of Fabry disease), Alidornase α (treatment of cystic fibrosis) and OPRX-106 (inflammatory bowel diseases) which have reached different clinical trials phases. Greenovation GmbH, has developed a moss-based platform in which Moss-aGal (treatment of Fabry disease) is under clinical trial phase I investigation, whereas Moss-FH (human complement Factor H) and Moss-GAA (treatment of Pompe disease) are in preclinical development

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