Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation

Abstract

Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, gamma-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera (gamma-Zein ER-accumulating domain) is the N-terminal proline-rich domain of gamma-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera domain. We demonstrated that Zera-F1-V protein accumulation was at least 3x higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera technology to induce protein body formation in non-seed tissues. Zera expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera technology to substantially increase the accumulation of value-added proteins in plants.

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

“…Previous work demonstrated that the proline-rich N-terminal domain of ␥-zein (Zera) fused to a protein induced PB formation in plant vegetative tissues (24,25,47).…”

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“…The Zera® domain, (N-terminal proline-rich domain: γZein ER-accumulating domain) of maize Zein gene is able to form PB bodies. This domain, in addition to other constituents, led to a threefold increase in the Yersinia pestis F1-V antigen fusion protein accumulation when using the fusion construct, in comparison to the construct lacking it, in three separate transient or stable transformation systems in Nicotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells (Alvarez et al, 2010). To facilitate the removal of these tags after purification, the recognition sequence of the protease TEV (the sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser)(ENLYFQ(G/S)) can be added between the protein of interest and the tag (Kapust & Waugh, 2000;Tropea, Cherry, & Waugh, 2009;Waugh, 2011).…”

“…However, the small tags in plant molecular pharming are characterized by their ineffectiveness, lack of scalability and high cost (Joensuu et al, 2010;Waugh, 2005). Some fusion tags have increasingly been gaining more attention as a new, effective way of facilitating the expression, extraction and purification of pharmaceutical proteins, such as hydrophobin (Joensuu et al, 2010;Joensuu, Conley, Linder, & Menassa, 2012), zein domain (Alvarez, Topal, Martin, & Cardineau, 2010), elastin-like polypeptides (Conley, Joensuu, Richman, & Menassa, 2011;Floss et al, 2009) and soybean agglutinin (SBA). The SBA affinity tag is an effective system which can be used for quick and efficient purification of recombinant proteins (Tremblay, et al 2011).…”

Transgenic crops for the production of recombinant vaccines and anti-microbial antibodies

“…44,45 A fusion protein comprising Yersinia pestis antigen F1-V and the proline-rich domain of γ-zein accumulates to threefold the levels of the F1-V antigen alone, through the induction of heterologous protein bodies. 43 The Spanish company ERA Biotech has commercialized this platform as Zera ® technology for the large-scale production of pharmaceutical proteins in plants.…”

“…42 An approach to achieve both goals at the same time is the induction of recombinant protein bodies containing the vaccine antigen. 43 Protein bodies are organelles derived from the ER; they occur naturally in seeds and are used to stockpile storage proteins. Evidence suggests that the storage proteins themselves contain structures that induce proteinbody formation: the proline-rich N-terminal domain of γ-zein, can induce protein bodies in cells that normally do not contain such co-accumulate with oleosin, allowing enrichment of the recombinant protein in the oil body fraction, which can be separated easily.…”