411

Recombinant proteins from transgenic plants Eva Franken, Ute Teuschel and Rtidiger Hain

Transgenic plants can express a wide variety of foreign genes

and offer the opportunity of large-scale protein production

in agricultural systems. The recombinant protein can serve

both ex situ and in situ purposes. Due to significant progress

in plant molecular biology, many different plant species can

now be transformed and are even capable of producing

very complex proteins such as antibodies or vaccines.

Furthermore, recombinant proteins can mediate resistance

against microbial pathogens, such as fungi or viruses, or

protect transgenic plants from insect pests.

Address

Bayer AG, Agrochemicals Division, Research/Biotechnology,

Landwirtschaftszentrum Monheim, 51368 Leverkusen, Germany

Current Opinion in Biotechnology 1997, 8:41 l-41 6

http://biomednet.com/ecref/0958166900800411

0 Current Biology Ud ISSN 0958-l 669

Abbreviations

MEV mink enteritus virus RIP ribosome-inactivating protein

SCFV single chain Fv fragment

Introduction During the past decade, fundamental changes have taken

place in the field of plant molecular biology [ 1.1. Together with improved techniques for plant genetic analysis and engineering, concepts of exploiting transgenic plants have gained increasing scientific and economic importance. Among the large number of new technologies that are available, commercial interest has focused on the ability of plants to integrate and express foreign genes and to produce recombinant proteins in bulk quantities at relatively low cost.

Plants now offer a number of expression systems for the temporally and spatially controlled production of recom- binant proteins for many different purposes (Figure 1). While molecular farming focuses on the utilization of plants or cultured plant cells as bioreactors producing the protein of interest for purification and further use (ex situ application), molecular breeding exploits transgenic protein expression that renders the plant of interest resistant against herbicides, insect or microbial attack (in situ application).

A large variety of plasmid constructs are used for the transformation, integration and expression of foreign genes in transgenic plants, including different promoter and targeting sequences. The most widely used technique for the transformation of plants is the Agrobacterium Ti-plasmid binary system [2,3], where the genes of interest are first introduced into Agrobacterium tumefaciens and plant

cells are then infected with the recombinant bacteria. The introduction of DNA into plant protoplasts via direct gene transfer using electroporation [4], polyethylene glycol mediated gene uptake [S], co-precipitation with calcium phosphate [6] or particle bombardment [7] facilitates the transformation of monocotyledonous plants, which are not readily infected by Agrobacterium strains.

Two major approaches have been made towards exploit- ing plant expression systems. Whole plants are used expressing the gene of interest and allowing recombinant protein production on an agronomical level. This strategy is especially useful in the case of proteins designated to improve the nutritional value of human or animal food plants, and for gene products causing plant resistance. In some special applications, the produced protein is directed to certain plant organs and, for example, accumulates in the fruits or seeds. On the other hand, plant cells cultured at high density in bioreactors allow the secretion of recombinant protein into the culture medium for better accessibility and easier purification.

Molecular farming Plantibodies

There are many examples of plants that produce trans- genie proteins serving a wide spectrum of purposes. An important possible application of plant expression systems originates from their ability to synthezise virtually every kind of antibody molecule. These ‘plantibodies’ can be isolated and used for ex situ purposes in diagnosis and therapy, or in situ, acting directly in the producing plant [8]. Complex antibodies consist of heavy and light chains covalently linked together by disulfide bonds. Each chain is folded into a series of variable (antigen-binding activ- ity) and constant (secondary effector function) domains. Transgenic plants use the same pathway for the assembly of light and heavy chains as mammalian cells involving similar signal peptides and chaperones for successful folding (see [9] for a review with regard to processing and engineering of plantibodies; it also discusses potential regulatory issues for therapeutic purposes). As in mam- malian cells, antibodies are secreted following assembly and post-translational processing. A difference seems to be in glycosylation patterns: plant antibodies lack N-acetyl neuraminic acid, which is the predominant terminal sugar residue in mammalian complex glycans. Single-chain Fv fragments (scFv) consist of only the variable light and variable heavy chain domains, which are covalently linked by a synthetic linker peptide (Figure 2). They bind to their antigens with similar affinities as the parental antibodies [lo].

Functional recombinant scFv fragments can be produced and even stored in cransgenic plants. As a model system,

412 Protein engineering

Figure 1

Insect

Viral

in situ

Production

Molecular breeding Molecular farming

Enzymes

Antibodies

Fields of application for transgenic plants. In situ, recombinant proteins protect plants from fungal, microbial, viral or insect attack. For ex situ purposes, recombinant proteins, such as enzymes or antibodies, can be produced by transgenic plants.

the legumin B4 gene promoter from l&z faba, which Ma et a/. [ 141 were the first to describe the production of a confers strict seed-specific expression, was used to express functional, high molecular weight secretory immunoglob- scFv in transgenic tobacco seeds [ 111. The transgenic scFv ulin in transgenic tobacco. Genes encoding the heavy was functional and had similar antigen binding affinity as and light chains of murine antibody (Guy’s 13 against an E. cob derived scFv and accounted for up to 0.67% of streptococcal antigen (SA) I/II), a murine joining chain, total soluble mature seed protein. These transgenic seeds and a rabbit secretory component were introduced into could be stored at room temperature for one year without separate transgenic tobacco plants. By cross-pollination, detectable loss of scFv content or activity. Increased plants were obtained that co-expressed all components expression levels of scFv (yielding up to 1% of the total and produced a functionally active secretory antibody that soluble protein) targeted to the cytosol or the secretory recognized the SA I/II cell surface adhesion molecule of pathway in transgenic tobacco were reported [12] when Stmptococcus mutans and Stmptococcus sobtitm. The anti- scFv was provided with the carboxy-terminal aminoacid body yield from fully expanded leaf lamina was 200-500 l.tg sequence KDEL, which is a signal for retention in the per gram of fresh weight material. Until then, complex endoplasmic reticulum. The affinity of plant-produced secretory monoclonal antibodies could only be generated scFv was about SO-fold lower than that of the parental by extremely complicated techniques like the in vitro antibody fragment when measured by enzyme linked conjugation of secreted dimeric immunoglobulin from immunosorbent assay. This could be due to the different mammalian plasma cells with the secretory component glycosylation patterns of plants and animals [13]. derived from epithelial cells. The large scale agronomic

Recombinant proteins from transgenic plants Franken, Teuschel and Hain 413

production of such functional secretory antibodies holds considerable potential for passive immunotherapy of mucosal surfaces, where large quantities of monoclonal antibodies are required [9].

Figure 2

Whole antibody

Single-chain Fv fragment

In antibodies, two heavy and two light chains are covalently linked together by interchain disulfide bonds (S-S). Both heavy and light chains contain variable regions (shown with bars) and constant regions. Single-chain Fv fragments consist of only the variable regions of a heavy and a light chain joined together by a synthetic polypeptide linker.

In addition to antibody production for ex situ tech- niques, in situ applications have become an important field of research. Although plants lack the mammalian antibody-triggered mechanism of antigen disposal, an- tibody binding can lead to agglutination of pathogens or block epitopes essential for infection. This effect is well documented for virus infection. Tobacco plants expressing a cytoplasmically targeted scFv against an epitope of the coat protein of artichoke mottled crinkle virus were less susceptible to infection with this virus [15]. Expression of a full-length heavy- and light-chain antibody against surface epitopes of the tobacco mosaic virus, possessing a signal peptide that targeted secretion to the extracellular compartment, mediated significant protection of transgenic tobacco plants [16]. More recent studies focus on the root-knot nematode Meloidogyne incognita. Transient expression assays with tobacco leaf protoplasts resulted in a high intracellular accumulation of functional scFv (of monoclonal antibody 6D4) directed against salivary secretions of this nematode, which are known to play a key role in the infection process (171. No influence on root-knot nematode parasitism could be observed following expression of functional full-length heavy and light chains of 6D4 in leaves, stems, roots, and galls of h-o&ma t&z~-urn cv. Xanthi tobacco (181. As a possible explanation, the apoplastic accumulation of the plantibodies is supposed, whereas nematode stylet secretions of saliva might be injected into the cytoplasm of the parasitized cell.

Other proteins and enzymes

Particular attention has been paid to the production of edible vaccines by expressing immunogenic viral proteins in transgenic plants [19]. The capsid protein of Norwalk virus, a virus causing epidemic acute gastroenteritis in humans, was shown to self-assemble into virus-like particles when expressed in transgenic tobacco and potato [ZO’]. Both purified virus-like particles and transgenic potato tubers stimulated the production of antibodies against Norwalk virus capsid protein when fed to mice. Dalsgaard et a/. [Zl’] used another approach for producing plant-derived vaccines. They inserted a DNA sequence coding for a short linear epitope from the VP2 capsid protein of mink enteritis virus (MEV) into an infectious cDNA clone of cowpea mosaic virus. When the construct was propagated in the black-eyed bean Vigna unguiculata, the MEV epitope was expressed on the surface of the recombinant cowpea mosaic virus, which successfully conferred protection against MEV after subcutaneous injection in mink.

With the aim of improving the nutritional status of agronomically important pasture legumes, a sulfur-rich seed albumin from sunflower was expressed in the leaves of transgenic subterranean clover [22]. By targeting the recombinant protein to the endoplasmic reticulum of the transgenic plant leaf cells, an accumulation of transgenic sunflower seed albumin of up to 1.3% of total extractable protein could be achieved.

Another approach exploits oleosins as carriers for foreign proteins. Oleosins are highly lipophilic proteins that accumulate on the surface of plant seed oil-bodies and are easily co-separated from the rest of the cellular extract by centrifugation. By constructing fusions with Arabidopsis oleosin genes it was possible to target recombinant protein, with approximately 80% of the activity partitioning with oil-bodies of Brassica napus [23]. The transgenic seeds could be stored for more than one year without significant loss of recombinant protein activity. Purification of the recombinant protein would involve endopeptidase cleavage of the fusion protein. Alternatively, for example, in the case of an enzyme, the oil-body could be used as an immobilization matrix. As a first example of this approach, hirudin was produced by exploiting oleosin partition- ing [24]. The oleosin-hirudin fusion protein accounted for up to 1% of the total seed protein and was biologically active after proteolytic release. Thus, exploiting oleosins as carriers for foreign proteins provides for easier purification and long-term storage of recombinant proteins.

Cultured plant cells In recent years, the production of high-value proteins by cultured plant cells has gained considerable attention. Cultured plant cells are especially suitable when the pro- tein of interest has to undergo complex post-translational processing, which cannot be accomplished by recombinant

414 Protein engineering

microbial systems and animal cell culture may be too costly. As intact transgenic plants require cultivation, harvesting, and extraction, recombinant protein production in plant cell cultures could provide significant economic advantage. Particularly high yields can be achieved in sys- tems where the recombinant protein does not inhibit cell growth by accumulating in the cells but is continuously secreted into the culture medium. Culture conditions can considerably influence the yield of recombinant protein. For example, by the addition of the protein-stabilizing agent polyvinylpyrrolidone to the culture medium, the level of secreted mouse monoclonal antibody heavy chain increased by 35fold to 0.36 pg protein/ml culture medium in suspension culture of transgenic tobacco cells [ZS]. The use of a perfusion air-lift bioreactor for high density Anchsa of/ha/is cell cultivation yielded cell densities of 27.2g/l dry weight and total extracellular protein concentrations of 1.1 g/l in contrast to only 12.6 g/l dry weight and 0.47gJ extracellular protein for batch culture [26*].

Molecular breeding In addition to recombinant protein production, gene expression in transgenic plants for in situ purposes can solve agricultural problems. This is particularly true when the expressed foreign protein enables important crop plants to cope with microbial or insect attack. There are a number of ways to achieve pest resistance by transgenic plants, involving a variety of naturally occurring resistance-mediating proteins, and more recently exploiting synthetic resistance genes.

In addition to the common approach of engineering virus resistance by expressing viral coat proteins, serveral other translatable viral sequences are currently being exploited as pathogen-derived resistance genes (reviewed in [27,28]). Other systems exploit transgenic plants ex- pressing naturally occurring antiviral genes. Trichosanthin, a ribosome-inactivating protein (RIP) from the Chinese medicinal herb Ttichosantfies kirilowii, accounted for up to 0.1% of the total soluble protein in transgenic tobacco leaves, and the plants were found to be completely resistant to mechanical inoculation of turnip mosaic virus [29]. In a similar approach, the RIP dianthin was inducibly expressed using the virion-sense promoter from African cassava mosaic virus in order to avoid the toxic effects of constitutive RIP expression on the transgenic plant. When inoculated with the African cassava mosaic virus, transgenic Nicotiana benthamiana plants exhibited attenuated systemic symptoms of virus infection, from which they recovered [30*]. Expression of the human antiviral RNA-decay-pathway enzymes Z-5A synthetase alone and RNase L alone was insufficient to protect transgenic tobacco plants. As demonstrated by activation experiments of RNase L in the transgenic plant leaves, both enzymes are required for antiviral activity [31].

Resistance of transgenic plants against fungal pathogens could be achieved by constitutively expressing enzymes

that can hydrolyze fungal cell walls, like chitinase [32], B-1,3-glucanase [33], or both [34]. Cysteine-rich antimicro- bial peptides from Mirabi/is jafapa and Amaranths caudatus were expressed in transgenic tobacco [35]; although they were functional in in vitro studies, they did not enhance plant resistance. Redirected targeting of the peptides from the extracellular spaces into an intracellular compartment also had no effect on the severity of the disease symptoms. A possible explanation for the ineffectiveness of the antimicrobial peptides to enhance plant resistance could be seen in their low level of expression or the sensitivity of these disulfide-linked peptides to the ionic strength of their enviroment.

Several approaches have been made towards insect resistance of important crop plants. The constitutive expression of cowpea trypsin inhibitor conferred on transgenic rice resistance against the yellow stem borer Scirpophaga incertuias and the striped stem borer ChiJo suppressah, the two major insect pests of rice in Asia [36]. While snowdrop lectin or trypsin inhibitor expressed in transgenic potato plants had a significant antifeedant effect on the tomato moth in growth room trials, bean chitinase was insufficient for plant protection [37]. Since the late eighties, the insecticidal crystal toxin genes from Bacillus thwingiensis have been used to engineer insect resistance in transgenic plants [38]; however, due to different codon usage by plants and bacteria, in most cases the level of expression was too low for efficient protection. Modifications of the toxin genes, such as removal of A+T-rich sequences that may cause mRNA instability or aberrant splicing, adjustment of the codon usage to make it more similar to that of the host plant, or truncation of the coding sequences have considerably increased toxin expression levels. Thus, the more recent approaches utilize modified synthetic crystal toxin genes for an effective insect pest control in rice [39], canola [40], soybean [41], alfalfa and tobacco [42]. Ten years after the first publication [43], transgenic B. t/lutingiensis-plants (e.g. Bollgard@ and Newleave@) have now been introduced into the US market.

Conclusions Molecular plant breeding is developing at a breath-taking rate. The first successful introduction of a fragment of foreign DNA into a plant cell in vitro was reported only 14 years ago [44]. Yet, the first products of this technology are already fully developed. Some transgenic products are already on the market, others are soon to be introduced. Regulatory considerations for the exploitation of transgenic plants will have to include their genetic stability, the purity of recombinant proteins, especially when they are determined for use in humans, and their environmental impact [45’]. Elaborated methods in plant molecular genetics allow the transformation of virtually every plant species and the expression of a large variety of genes. Thanks to these new technologies, we now have

Recombinant proteins from transgenic plants Franken, Teuschel and Hain 415

the ability to use transgenic plants cultivated on an agricul- tural scale as potentially safe and cost-effective sources to produce almost limitless amounts of recombinant protein.

References and recommended reading Papers of Particular interest, published within the annual period of review, have been highlighted as:

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This IS an tnterestlng paper tor sclentlsts who are conslaerlng tne practlcar application of their research. The author discusses practical problems like euhty or product comparability as well as environmental concerns. For the United States, he provides some addresses where guidance documents can be obtained.