Seed‐based expression systems for plant molecular farming

Abstract

SummaryThe evolution of the seed system provides enormous adaptability to the gymnosperms and angiosperms, because of the properties of dormancy, nutrient storage and seedling vigour. Many of the unique properties of seeds can be exploited in molecular farming applications, particularly where it is desirable to produce large quantities of a recombinant protein. Seeds of transgenic plants have been widely used to generate a raw material for the extraction and isolation of proteins and polypeptides, which can be processed into valuable biopharmaceuticals. The factors that control high-level accumulation of recombinant proteins in seed are reviewed in the following paragraphs. These include promoters and enhancers, which regulate transcript abundance. However, it is shown that subcellular trafficking and targeting of the desired polypeptides or proteins play a crucial role in their accumulation at economically useful levels. Seeds have proven to be versatile hosts for recombinant proteins of all types, including peptides or short and long polypeptides as well as complex, noncontiguous proteins like antibodies and other immunoglobulins. The extraction and recovery of recombinant proteins from seeds is greatly assisted by their dormancy properties, because this allows for long-term stability of stored products including recombinant proteins and a decoupling of processing from the growth and harvest cycles. Furthermore, the low water content and relatively low bioload of seeds help greatly in designing cost-effective manufacturing processes for the desired active pharmaceutical ingredient. The development of cGMP processes based on seed-derived materials has only been attempted by a few groups to date, but we provide a review of the key issues and criteria based on interactions with Food and Drug Administration and European Medicines Agency. This article uses 'case studies' to highlight the utility of seeds as vehicles for pharmaceutical production including: insulin, human growth hormone, lysozyme and lactoferrin. These examples serve to illustrate the preclinical and, in one case, clinical information required to move these plant-derived molecules through the research phase and into the regulatory pathway en route to eventual approval.

Large-scale production of functional human serum albumin from transgenic rice seeds

“…In general, downstream processing of recombinant pharmaceutical proteins are cost-consuming for many bioreactor systems, including those that use plants (23,32). Our protocol for purification of OsrHSA is robust and economical, even at the kilogram scale.…”

“…Plant seeds, especially cereal crop seeds, are promising vehicles for producing recombinant proteins, because they can achieve high accumulation of recombinant protein, display high levels of protein stability, stored for long periods of time, and are well controlled on a production scale (22,23). Human lysozyme and lactoferrin produced for oral administration have been successfully expressed in rice seeds (24,25).…”

Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants

“…We used total chloroplasts as the starting material in the purification procedure, because of the association of PGL35-HIVp24 with both the plastoglobule and thylakoid fractions. For this reason also, purification protocols resembling the one in the oleosin system (Vanrooijen and Moloney 1995;Boothe et al 2010) using a flotation centrifugation step may be inefficient for most plastoglobulin fusion proteins. It is possible however that PGL35 fusion proteins that are more hydrophobic and have a lower isoelectric point may accumulate to a high degree in plastoglobules and be more readily purified by flotation centrifugation.…”

Transgenic Expression and Identification of Recombinant Human Proinsulin in Peanut

“…From 2004 to 2013, biopharmaceuticals were largely derived from E. Coli (24%), yeast (13%), mammalian cells (56%), transgenic animals and plant expression systems (3%) and insect cells (4%) (Gurramkonda et al 2010;Qian et al 2011;Walsh 2012;Nielsen 2013;Walsh 2013;Baeshen et al 2014). Transgenic plant expression systems have attracted attention due to advantages such as high-capacity production, safety, inexpensive investment, and fast and easy scale-up (Arakawa et al 1998;Ruhlman et al 2007;Xie et al 2008;Boothe et al 2010;Boyhan and Daniell 2011;Soltanmohammadi et al 2014). Transgenic seeds and leaves exhibit long-term stability and can be used to stockpile insulin until it is needed.…”

“…Peanut seeds are rich in protein and fat and can serve as a good reservoir for heterologously expressed proteins; thus, we sought to express the human insulin gene in peanut seeds. Because insulin is highly unstable and is prone to Nterminal degradation (Boothe et al 2010), oleosin or flag tag was fused with proinsulin to protect the Nterminus from degradation. Otherwise the recombination protein purification cost will be reduced.…”