Plants are gaining acceptance as a platform for large-scale production of recombinant proteins. Over the past 10 years, several efficient plant-based expression systems have emerged. The main advantage is the anticipated cost savings, reflecting the large amount of biomass that can be produced in a short time with no need for specialized equipment or expensive media.
Sowing, growing and harvesting can be carried out using traditional agricultural practices and unskilled labor. In addition there's no danger for contamination with human pathogens such as viruses and prions, or oncogenic DNA sequences as is the case for mammalian cell cultures or bacterial endotoxins. Overcoming bottlenecks imposed by low yields, poor and inconsistent product quality and difficulties with downstream processing are the most important goals for researchers working in this field. Strategies to ameliorate recombinant protein yield in plants include development of better expression cassettes, improvement of protein stability and accumulation by using specific subcellular targeting signals, and development of downstream processing technologies.
In this perspective, seed based platforms are particularly attractive because they allow recombinant proteins to stably accumulate for long periods of time at a relatively high concentration in a compact biomass in which they are protected from degradation, which is beneficial for extraction and downstream processing. Indeed, 20-40% of the total seed content consists of protein, while for leaves this is only 2%. This implicates that the amount of recombinant protein per gram seed is much higher in seeds than in leaves.
Given these advantages, our research is focused on the use of non-food dicotyledonous species for the production of recombinant proteins, using Arabidopsis as a model. By using a seed-specific expression cassette based on the regulatory signals of seed storage proteins of common bean (Phaseolus vulgaris), and by targeting the recombinant protein to the endoplasmatic reticulum (ER), we obtained the highest yields of recombinant proteins in plants described so far: a single-chain variable fragment (scFv) accumulated to levels in excess of 36% of total soluble protein (TSP) in homozygous Arabidopsis seeds, while retaining its antigen-binding activity and affinity (De Jaeger et al., 2002). The major advantage of Arabidopsis seeds as production platform for important and high value proteins is the small size of the plant and consequently the small amount of space needed to grow a large amount of plants. Furthermore, Arabidopsis plants can easily grow in greenhouse conditions.
However, despite the fact that seeds are convenient tissues in which to express recombinant proteins, heterologous expression has been studied primarily in leaves, and much less is known about production in seeds. Recent studies indicate that factors specific to the plant species, tissue and physiological state can have a significant impact on the amount and quality of the recombinant product. More detailed comparative studies are therefore needed for each protein, including the analysis of expression levels, biochemical properties, subcellular localization and in vitro and in vivo activity.
Research therefore focuses on the following topics:
Loos et al. (2010) Production of monoclonal antibodies with a controlled N-glycosylation pattern in seeds of Arabidopsis thaliana. Article first published online: 14 JUN 2010. DOI: 10.1111/j.1467-7652.2010.00540.x
Van Droogenbroeck et al. (2007) Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds. PNAS 104:1430-1435.
Eeckhout et al. (2004) A technology platform for the fast production of monoclonal recombinant antibodies against plant proteins and peptides. J. Immunol. Methods 294: 181-187.
De Jaeger et al. (2002) Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences. Nature Biotechnol. 20: 1265-1268.
Peeters et al. (2001) Production of antibodies and antibody fragments in plants. Vaccine 19: 2756-2761.
Post-docs: Sylvie De Buck, Annelies De Paepe
Technicians: Jonah Nolf, Els Van Lerberge
PhD students: Thomas De Meyer, Kirsten De Wilde, Miguel Lopez Cardoso, Robin Piron, Vikram Virdi
Undergraduate student: Gert-Jan Van Landeghem