Bio-pharmaceuticals and technical enzymesexpressed in
1. Introduction
The
The
The purification of heterologous products can be a costly process, although in some situations this step is not needed; a good example is provided by the feeding to poultry of transgenic pea expressing an scFv antibody directed against the
To date, the main focus of pharma transgenic research in the
Uniform planting material is a necessity, and one means of obtaining this in the cereals is to generate doubled haploid plants from immature pollen. In barley, Kumlehn et al. (2006) were able byusing
This review aims to summarize the current state of the art regarding strategies, targets and future challenges in order to achieve high expression levels of
2. The generation of transgenic plants
The progress achieved over the past 20 years towards
2.1. Biolistic gene transfer
The biolistic technique involves the bombardment of the recipient tissue with gold or tungsten particles coated with the transgene DNA. It has been widely used to achieve transient expression, particularly where the purpose has been to assess the functionality of gene candidates, the effectiveness of RNAi constructs or the activity of promoter/reporter fusions (Onate et al., 1999; Rubio-Somoza et al., 2006). The major advantage of the technique is that it can rapidly characterize a large number of sequences (Ihlow et al., 2008). Most biolistic protocols seek to effect transfer into either leaf epidermal cells (Douchkov et al., 2005) or into the scutellar tissue of an immature embryo (Knudsen & Müller, 1991). The first stable transgenic wheat plants generated by this means involved the introduction of a gene determining herbicide resistance into embryogenic callus (Vasil et al., 1992). Thereafter, the method was improved and applied successfully to barley (Wan & Lemaux, 1994), cereal rye (Castillo et al., 1994), triticale (Zimny et al., 1995) and macaroni wheat (Bommineni et al., 1997).
2.2. Agrobacterium -mediated gene transfer
Although
3. Expression systems
A comprehensive summary of the expression systems developed to date has been given by Hensel et al. (2011). In the context of the cereal grain, a prime target has been to exploit the regulatory system responsible for the expression of the endosperm storage proteins, which represent a major proportion of the protein synthesized within the grain. A particularly frequently exploited sequence for barley is the
4. Targets
Three major groups of products have been targeted to date for molecular farming. The first two consist of human or animal antigens and antibodies, which have applications in disease diagnosis, prophylaxis and recovery. The third, which has reached a more advanced stage thanks to a lesser regulatory load, is a range of technical enzymes. The first plant-made protein to be marketed was chicken avidin, produced in maize by ProdiGene (Hood et al., 1997). A number of companies have been active in making recombinant proteins in
Barley | Enterotoxigenic | Edible vaccine for pigs partialy effective against ETEC- induced diarrhea | Barley | Joensuu et al., 2006 |
Maize | ScFvT84.66 | Antibody against carcinoembryonic antigen (CEA), tumor-associated diagnostic reagent | Wheat | Stoeger et al., 2000 |
Wheat High-molecular-weight | Synthetic anti glycophorin scFv-HIV epitope fusion | HIV diagnostic reagent | Barley | Schuenmann et al., 2002 |
Barley | Molecular farming of pharmaceutical proteins | Barley | Stahl et al., 2002 | |
Barley | ||||
Barley | Molecular farming of pharmaceutical proteins | Barley | Stahl et al., 2002 | |
Barley | Molecular farming of pharmaceutical proteins | Barley | Stahl et al., 2002 | |
Maize | Molecular farming of pharmaceutical proteins | Barley | Ritala et al., 2008; Eskelin et al., 2009 | |
Rice | ||||
Barley | Molecular farming of pharmaceutical proteins | Barley | Erlendsson et al., 2010 | |
Barley | Molecular farming of pharmaceutical proteins | Barley | Stahl et al., 2002 | |
Maize | Kamenarova et al., 2007 | |||
Rice | ||||
Barley | Molecular farming of pharmaceutical proteins | Barley | Stahl et al., 2002 | |
Rice | Huang et al., 2006 | |||
Wheat High-molecular-weight | Wheat | Huang et al., 2010 | ||
Barley | Molecular farming of pharmaceutical proteins | Barley | ORF Genetics | |
Wheat High-molecular-weight | An- | Molecular farming of second generation biofuels | Wheat | Harholt et al., 2010 |
Barley | Heat stable | Grains containing thermostable 1,3-1,4- β-glucanase for better malting | Barley | Horvath et al., 2000 |
Maize | Grains with altered oxygen availability | Barley | Wilhelmson et al., 2007 | |
Wheat Low-molecular-weight | Ps- | Grains with altered protein composition | Wheat | Stoeger et al., 2001 |
Hv- | Plants with modified oxylipin signature | Barley | Sharma et al., 2006 | |
Maize | Heat-stable An- | Grains with improved digestibility for non-ruminant animal feed | Wheat | Brinch-Pederson et al., 2000 |
Barley | Td- | Grains containing a natural sweetener for brewing industry | Barley | Stahl et al., 2009 |
Wheat High-molecular-weight | Bs- | Grains with improved baking quality | Wheat | Harholt et al., 2010 |
4.1. Vaccines andantigens
Epidemics of the major infectious human diseases are becoming rare in the developed world thanks to the widespread use of vaccination. In less developed countries, the high cost of vaccine and a poorer level of social infrastructure exposes the population to such diseases. The production of a cheap prophylactic product, such as a plant-made vaccine, would make a material contribution to development. The ideal expression system for producing such vaccines needs to be readily transformable, inherently safe and economical, and therapeutically effective (Fischer and Schillberg, 2004). Current systems capable of producing antigens and antibodies in transgenic plants have recently been described (Daniell et al., 2009; Floss et al., 2009; Joensuu et al., 2008). While vaccines can be administered either orally or by injection, the former method is preferably from an organizational point of view and the use of grains (or other plant parts) is particularly attractive for the vaccination of domesticated animals. A disadvantage of the oral delivery route is the relatively large quantity of antigen required (Streatfield & Howard, 2003). The only published report which describes the use of
4.2. Antibodies
Following the first discovery of immunity-conferring substances in the blood (Behring & Kitasato, 1890), antibodies have been exploited in the fight against several diseases. Most antibodies are large Y-shaped proteins that include an antigen-binding site formed by the two variable segments of their heavy and light chain. The five major classes of antibody (IgA, IgD, IgE, IgG and IgM) are recognized by their conserved region structure and their immunological function (Woof & Burton, 2004). Hiatt et al. (1989) pioneered the expression of immunoglobulin chains in tobacco, since then, various portions of these chains have been expressed heterologously, including single chain molecules (scFvs), Fab fragments, small immune proteins (SIPs), IgGs and chimeric secretory IgAs (for a review, see De Muynck et al., 2010). The commonest plant host to date has been tobacco, with only a small number of examples among the
4.3. Human proteins and growth factors
The earliest published account of the use of cereal grain to express human genes concerned the five proteins antithrombin III, α1-antitrypsin, lysozyme, serum albumin and lactoferrin (Stahl et al., 2002). Here, the concern was not the quantity or quality of the recombinant proteins, but rather the detection of the T-DNA integration sites in the barley genome. However, these targets remain in the portfolio of Maltagen Forschung GmbH, whose website provides detailed information concerning the company's interest in these genes (Maltagen, Germany). Similar products are also offered by ORF Genetics, which exploits an endosperm-specific expression system. They produce a number of hormones and cytokinines like endothelial monocyte activating polypeptide-2 (EMAP2), various fibroblast growth factors, interferons and interleukins. A recent product from this company was human FLT3-ligand, with the gene under the control of the barley
4.4. Technical enzymes and recombinant proteins
Here, the focus wason transgenes whose products are designed to either improve the technical quality of wheat (baking) or barley (brewing), to alter feed quality, or to improve biofuel properties. The earliest report of this sort of manipulation dates back about a decade, when Horvath et al. (2000) described the heterologous expression of a gene encoding a heat-stable
Barley malt and wheat flour are common ingredients of processed food and beverages, so the improvement of their technical quality is of commercial interest. The protein thaumatin is a low-calorie sweetener and flavour modifier(Gibbs et al., 1996;Green, 1999), initially isolated from the West African katemfe fruit (
The germinating seed frequently suffers from oxygen deficiency (Bewley & Black, 1994). This presents a problem during the malting process, and is not readily counteracted by continuous aeration (Wilhelmson et al., 2006). The hypoxia inhibits the
Several studies have highlighted the role of oxylipins in the regulation of environmentally induced or developmental-specific processes (Weber, 2002). Oxylipins are a product of the lipoxygenase pathway. When barley
The baking property of wheat flour is influenced largely by the quantity and quality of the endosperm storage proteins, but arabinoxylan, the major non-starch polysaccharide present in the flour, also has some influence. When Harholt et al. (2010) created transgenic wheat plants expressing an
The major classes of endosperm storage proteins in the
5. Proteinmodifications
Several modifications occur during the processing of proteins; these include cleavage of signal peptides after entry into the ER, formation of disulphide bonds in the lumen of the rough ER, phosphorylation by protein kinases, and the attachment of sugar side chains (glycosylation) initiated in the ER but occurring primarily in the Golgi apparatus. These modifications can be an important determinant of a protein's stability and activity.
5.1. Disulfid bridges
The conformation of a protein is sequence-dependent. One of the primary determinants of folding is the formation of a disulphide bridge between pairs of thiol groups. Most prolamins contain a number of cysteine residues capable of forming such disulphide bonds. The retention of a phaseolin γ-zein fusion protein in the ER of tobacco protoplasts was shown to be dependent on disulphide bonding (Pompa & Vitale, 2006). Prolamins are synthesized in the ER of the wheat and barley endosperm, and are then transported to protein storage vacuoles (PSVs) in a process thought to involve both Golgi-dependent and independent pathways (Galili et al., 1993; Levanony et al., 1992; Rechinger et al., 1993). Autophagy and the
5.2. Glycosylation
More than 50% of eukaryotic proteins are glycosylated (Apweiler et al., 1999), with the sugar linked either to an asparagine (
6. Concluding remarks
This review has set out to summarize the information in the public domain regarding the use of
A number of challenges remain before plant-made pharmeceuticals (PMPs) can reach the market. A major one is the expense and low efficiency of target purification. The attachment of fungal hydrophobins, elastin-like polypeptides (ELPs) or the use of a domain of the maize storage protein zein as a purification tag represents promising strategies. The principle behind these purification tagging approaches can be based on either a temperature dependent change in solubility (ELP) termed inverse transition cycling (Meyer &Chilkoti, 1999), on a change in hydrophobicity in the case of the hydrophobins (Linder et al., 2001), or on the assembly of the proteins into so-called protein bodies by the use of γ-zein (Coleman et al., 1996; Geli et al., 1994). Although inverse transition cycling has been used to purify cytokines (Lin et al., 2006), antibodies (Floss et al., 2009; Joensuu et al., 2009) and spider silk proteins (Scheller et al., 2004) from transgenic plants, no application has yet been reported in
A more inexpensive approach is possible where the whole seed (or grain) is a component of feed, since in this case no purification is necessary. Nevertheless it remains important that the PMP is stable under ambient temperature conditions for several weeks. The stability of an antibody in the wheat grain was already demonstrated a decade ago (Stoger et al., 2001). Where the PMP is heat stable, then heat treatment during feed processing is possible (Horvath et al., 2000). Achieving an adequate level of expression is essential, one approach would be to lower the amount of endogenous storage proteins competing with the transgene. Such a strategy has been followed by ORF Genetics by the down regulation of an transcription factor (
Public acceptance of GM products and a straightforward means of their detection require the availability of clear markers. In barley it is possible to use testa colour for this purpose by conventionally transferring an exotic testa colour into a readily transformable cultivar, which then becomes suitable for the production of PMPs (Orvar, 2006). With the imminent acquisition of the genomic sequences of barley and wheat, it can be expected that the key genes for the synthesis and processing underlying the pattern of glycosylation of
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