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Transgenic Research 9: 279–299, 2000.

© 2000 Kluwer Academic Publishers. Printed in the Netherlands.

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Molecular farming of pharmaceutical proteins
Rainer Fischer1,2,∗ & Neil Emans1
1 Institut

für Biologie I (Botanik/Molekulargenetik), RWTH Aachen, Worringerweg 1, D-52074 Aachen, Germany
Department for Molecular Biotechnology, IUCT, Grafschaft, Auf dem Aberg 1, D-57392
Schmallenberg, Germany
2 Fraunhofer

Key words: molecular farming, recombinant protein, transgenic plant, antibody, expression, purification,
biotechnology

Abstract
Molecular farming is the production of pharmaceutically important and commercially valuable proteins in plants.
Its purpose is to provide a safe and inexpensive means for the mass production of recombinant pharmaceutical
proteins. Complex mammalian proteins can be produced in transformed plants or transformed plant suspension
cells. Plants are suitable for the production of pharmaceutical proteins on a field scale because the expressed
proteins are functional and almost indistinguishable from their mammalian counterparts. The breadth of therapeutic
proteins produced by plants range from interleukins to recombinant antibodies. Molecular farming in plants has
the potential to provide virtually unlimited quantities of recombinant proteins for use as diagnostic and therapeutic
tools in health care and the life sciences. Plants produce a large amount of biomass and protein production can
be increased using plant suspension cell culture in fermenters, or by the propagation of stably transformed plant
lines in the field. Transgenic plants can also produce organs rich in a recombinant protein for its long-term storage.
This demonstrates the promise of using transgenic plants as bioreactors for the molecular farming of recombinant
therapeutics, including vaccines, diagnostics, such as recombinant antibodies, plasma proteins, cytokines and
growth factors.

Introduction
The use of plants in medicine stretches back to the
earliest stages of civilization. As early as 1600 BC, the
Egyptians compiled a list of more than 700 medicinal
plants. The active ingredients in many of these plants
have now been identified, and close to one quarter of
prescription drugs are still of plant origin. Modern biotechnology is extending the use of plants in medicine
well beyond its original boundaries. Plants are now a
source of pharmaceutical proteins, such as mammalian
antibodies (Hiatt et al., 1989; Düring et al., 1990;
Hiatt, 1990; Whitelam et al., 1994; Ma et al., 1995;
Conrad & Fiedler, 1998; Larrick et al., 1998; Zeitlin
et al., 1998; Fischer et al., 1999a), blood substitutes
(Magnuson et al., 1998) and vaccines (Arakawa et al.,
∗ Author for correspondence: Institut für Biologie I Tel.: +49
241 806631; Fax: +49 241 871062; E-mail: fischer@bio1.rwthaachen.de

1998a; Haq et al., 1995; Kapusta et al., 1999; Mason
& Arntzen, 1995; Mason et al., 1992; McGarvey et al.,
1995; Walmsley & Arntzen, 2000).
Molecular farming is the production of pharmaceutically important and commercially valuable proteins in plants (Franken et al., 1997). It harnesses heterologous protein expression systems, such as plants,
for the large-scale production of recombinant proteins
that are therapeutically valuable. Here, we briefly
review its history, then discuss plant expression technology and how plant cells can be optimally used to
produce recombinant proteins by molecular farming.
In its simplest form, molecular farming is the expression of recombinant insulin in bacteria; in its most
challenging form, molecular farming is the production
of chimeric anti-tumor antibodies and multi-subunit
protein complexes, like secretory IgA, in plants. This
technology has now reached the point where it is commercially viable. Its further development will bring

280
many therapies that are now too expensive for wide
use into the hands of most medical practitioners. We
anticipate that molecular farming may be a major area
of economic growth in agricultural biotechnology.
In our definition, molecular farming encompasses
the production of recombinant proteins in a heterologous expression system. In general, the recombinant
proteins that we choose to express are pharmaceutically valuable, such as a tumor or pathogen specific
antibody. Consequently, the expression of an antibody
in tobacco is molecular farming while the purification
of that antibody from its native source is not. Molecular farming a protein also implies that large-scale
production of a recombinant protein is both possible
and economically feasible. Thus, production of an
antibody in a field of transgenic tobacco is closer to
molecular farming than the production of the antibody
in a hybridoma culture. Molecular farming is principally the production of a pharmaceutical protein and
not the modification of the expression system. For
example, the creation of a pathogen resistant plant
by the expression of an anti-pathogen antibody is
only an application of the molecular farming technology, but it is not molecular farming according to our
definition.
Molecular farming is thus best defined as the expression of pharmaceutically and commercially valuable proteins in plants. It was developed based on
the pretext that many pharmaceutically active proteins
have been identified but without the means to produce
them, their therapeutic potential can not be evaluated.
The purpose of molecular farming is to produce large
amounts of an active, safe pharmaceutical protein at
an affordable price. As such, there are two stages to
molecular farming – the development of an optimized expression system and its scale up to economic
levels of production. We feel that plants are the expression system that best meets these prerequisites.
Further developments in protein expression in plants
should make molecular farming an even more attractive alternative to animals, animal cells and microbial
cells.
In this review, after a brief historical overview of
the development of molecular farming, we concentrate on discussing the expression of pharmaceutical
proteins in plants through stable or transient expression in whole plants, leaves or seeds. The economic
and practical advantages of plants are discussed, as is
where molecular farming is likely to expand. We then
speculate on how the use of plants as an expression
system may be improved.

The demand for safe, recombinant pharmaceutical proteins is expanding rapidly. Both the amounts
of proteins needed and protein complexity are increasing as novel pharmaceutical activities are identified or designed into macromolecules. We foresee
that molecular farming in plants will become a preeminent method for the production of pharmaceutical
molecules in the next 10 years.

Molecular farming
Molecular farming is the production of recombinant
pharmaceuticals outside their natural source. By definition, molecular farming is preceded by identification
of a protein with a desirable therapeutic or diagnostic
activity, its protein and DNA sequencing and finally its
expression in a heterologous host. A classic example
of molecular farming in microbes is the expression of
recombinant insulin in bacteria.
The anti-diabetic activity of insulin was first identified in 1921 and by 1951 the complete amino acid
sequence had been determined. Since the standard
source of the hormone was animal pancreas, a demand
emerged for an alternative source of insulin that was
safe, free of immunogenic contaminants and inexpensive. Human insulin was an attractive target for expression in microbes for it is a small polypeptide requiring
only minimal post-translational processing to become
functional. Expression in bacteria was successful and
in 1982 it became the first recombinant protein to
be approved for therapeutic use (Walsh, 1998). This
released any restrictions on the amounts of insulin
available, by producing a safe, active, recombinant
human hormone at a low cost.
The potential of using plants as a production system for recombinant pharmaceuticals was established
between 1986 and 1990 with the successful expression
of a human growth hormone fusion protein, an interferon and human serum albumin (Barta et al., 1986;
De Zoeten et al., 1989; Sijmons et al., 1990). A crucial advance came with the successful expression of
functional antibodies in plants in 1989 (Hiatt et al.,
1989) and 1990 (Düring et al., 1990). This was a
significant breakthrough for it showed that plants had
the potential to produce complex mammalian proteins
of medical importance. By analogy to the production
of insulin in bacteria, the production of antibodies
in plants had the potential to make large amounts
of safe, inexpensive antibodies available. This was
an impressive result because plants could produce

Ziegler et al. Contrastingly.. 1995. Ab – Antibody. 1995... indicating that all the post-translational modifications necessary for antibody activity occurred.. secretory IgA. plants were shown to be able to produce a variety of antibody fragments (Figure 1). Firstly. as a cheap. 1989. Voss et al.. dAb – single domain antibody. 2000) and plantderived antibodies are functionally equivalent to those produced by hybridomas (Hiatt et al. functional full-length antibodies. 1995). 1999).. scFv – single chain antibody fragment. proteins produced in plants accumulate to high levels (Verwoerd et al. blood substitutes and biological effectors including interleukins (Tables 2–4). 1995) and further progress has made it possible to produce chimeric mouse–human therapeutic antibodies in plants in sufficient quantities for pre-clinical trials (Zeitlin et al.281 Figure 1.. In the following 10 years. bacteria cannot produce full size antibodies nor perform most of the important mammalian post-translational modifications... Fab – fragment antigen binding. Advances in recombinant DNA technology. Thirdly. Ma et al. This limitation and the cost of expression of proteins in mammalian cells prompted the exploration of plants. Plant expression systems are attractive because they offer significant advantages over the classical expression systems based on bacterial. safe and efficient alternative. they are incapable of most of the post-translational modifications necessary for the activity of many mammalian proteins. 1994). Taticek et al. rAb – recombinant antibody. Forms of recombinant antibodies produced by antibody engineering. convenient production system. which is not the case for full size antibody expression in E. Voss et al. IL-2 – interleukin-2. they have a higher eukaryote protein synthesis pathway.. 1989. Antibody expression in plants showed proof that plants were capable of expressing functional mammalian proteins (Hiatt et al. concerns about contamination . plant transformation technology and antibody engineering are major reasons why plants have emerged as an expression system. Secondly. 1998. Plants as a production system Historically. bacteria were often the protein expression system of choice and yeast cells or baculovirusinfected insect cell systems were of minor importance (Skerra. very similar to animal cells with only minor differences in protein glycosylation (Cabanes-Macheteau et al. Vaquero et al. microbial and animal cells (Table 1). 1999). While bacteria are an inexpensive. 1993. coli..

Walmsley & Arntzen.. 1990. Current applications of the plant based expression systems in biotechnology include the production of recombinant antibodies (rAbs) (Ma and Hein. For example. Whitelam et al. In addition. 1993. Chimeric plant viruses. hormones. Mammalian cell cultivation can be difficult. enzymes (Hogue et al.282 Table 1. Bacteria produce contaminating endotoxins that are difficult to remove and bacterially expressed recombinant proteins often form inclusion bodies. Voss et al.. Classical methods of protein expression often require a significant investment in recombinant protein purification (bacteria) or require expensive growth media (animal cells). interleukins (Magnuson et al. expensive fermenters etc. requires sophisticated equipment and expensive media supplements. such as foetal calf serum. Verwoerd et al. is a great advantage over the more commonly used microbial methods. Transgenic plants producing high levels of safe. 1994. can also be used for the presentation of vaccines on the viral surface (Johnson et al. 1995. endotoxins.. of expressed proteins with human or animal pathogens (HIV. The ease with which plants can be genetically manipulated. functional recombinant proteins. Whitelam. plasma proteins (Sijmons et al. mineral salts and sunlight. Fecker . are entirely avoided by using plants. 1995a.. 1996) as a source of recombinant antibodies is becoming limited by legal and ethical constraints. pathogen resistance can be increased by the expression of anti-pathogen antibodies (Tavladoraki et al. 2000). making labour. Importantly. 1993. ∗∗ – large. considerable care must be also taken during downstream processing of recombinant proteins to remove oncogenic sequences.. the use of transgenic animals (Echelard. 1997). can be cultivated on an agricultural scale (Whitelam et al. Comparison of features of recombinant protein production in plants.. 1995. yeast and classical systems Cost/storage Distribution Gene size Glycosylation Multimeric protein assembly (SIgA) Production cost Production scale Production vehicle Propagation Protein folding accuracy Protein homogeneity Protein yield Public perception of ‘risk’ Safety Scale up costs Therapeutic risk∗ Time required Transgenic plants Plant viruses Yeast Bacteria Mammalian cell cultures Transgenic animals Cheap/RT Easy Not limited ‘Correct’ ? Yes Cheap/−20◦ C Easy Limited ‘Correct’ ? No Cheap/−20◦ C Feasible Unknown Incorrect No Cheap/−20◦ C Feasible Unknown Absent No expensive/N2 Difficult Limited ‘Correct’ No Expensive Difficult Limited ‘Correct’ Yes Low Worldwide Yes Easy High ? Low Worldwide Yes Feasible High ? Medium Limited Yes Easy Medium Medium Limited Yes Easy Low High Limited Yes Hard High High Limited Yes Feasible high High ? High High Medium Very high High Medium High Medium Low Medium Low Medium Medium-high Medium Low high High Unknown High∗∗ Low High∗∗ Medium High∗∗ High High Unknown Medium Yes Low Yes High Yes High High High Low Low (unlimited biomass) Unknown Unknown Medium Low ∗ – residual viral sequences. 1998)... water. antibody expression can also be used as a tool to modify the intrinsic properties of plants. 1990) and vaccines (Mason & Arntzen.. hepatitis viruses) or the co-purification of blood-borne pathogens and oncogenic sequences. With these classical expression systems.b). oncogenes. produced in plants.and cost-intensive in vitro refolding necessary. mammalian cell culture and transgenic animal technology. and grown in single cell suspension culture or scaled up for field scale production. ? – unclear. 1996) and molecular farming requires only a virus-infected or a transgenic plant. 1995). protein or viral contaminants for in vivo therapeutic applications.

tabacum seeds N. 1993) (Bosch et al. Plant expression strategies Plant transformation involves the chromosomal integration of a heterologous gene and this is becoming straightforward. tabacum (Dieryck et al. annus N.. Protein expression studies have demonstrated that many forms of recombinant antibody fragments (Figure 1) can be functionally expressed in plants and that the sub-cellular targeting of the protein is an important consideration for high level expression. tabacum H... human protein C serum protease Human α and β haemoglobin Human muscarinic cholinergic receptors Murine granulocyte-macrophage colony stimulating factor Interleukin-2 and Interleukin-4 N. 1994) (Parmenter et al. Selected pharmaceutical proteins expressed in transgenic plants Year Protein Transformed species Reference 1986 Human growth hormone (Barta et al. 1997). 2000) than some classical expression systems. tabacum Suspension cells N. or plant organs. sativa N. Developing plant lines expressing recombinant proteins is time intensive and expensive.. 1999) (Leite et al. tabacum N. 1998).. 1997) N. However. tabacum S. 1986) 1990 Human serum albumin 1993 1994 1994 1995 Human epidermal growth factor Trout growth factor Human α-interferon Hirudin 1995 Erythropoetin 1996 Glucocerebrosidase. tabacum (Lee et al. 8–12 weeks are needed for transgenic plants to be available.. tabacum Suspension cells N. In the best case. 1995) (Matsumoto et al.. whole plants.. sativa Suspension cells N. 2000) (Staub et al. such as developing efficient transformation techniques for all major crop species. tabacum O... Though this is slower (Sijmons et al... 1995) (Cramer et al. 1997) (Mu et al. Le Gall et al..283 Table 2.. there are still technical and logistical hurdles to be overcome. 1990) (Higo et al. Transient gene expression in plants Transient gene expression in plants has several advantages over the generation of stably transformed trans- . the development of transient expression systems (Kapila et al. 1999) (Terashima et al. tabacum Chloroplasts (Magnuson et al. but the time required depends on the plant species. tuberosum N... tabacum Rhizosecretion O. These observations seem to be applicable to both transient and stable expression in plants and whether the protein is expressed in suspension cells. 1997) N. 1996) (Borisjuk et al.. 1998. 1996) means that the expression vectors and protein levels achieved can be checked before making this investment.. tabacum N. Expressed antibodies can also be used to alter metabolic or hormonally regulated pathways by binding to intracellular substrates or hormones (Phillips et al.. tabacum Suspension cells N. 1994) (Zhu et al. tabacum N. Zimmermann et al... 1998) 1997 1997 1997 1998 1999 1999 2000 2000 Human placental alkaline phosphatase Human α1-antitrypsin Human growth hormone (somatotrophin) Human growth hormone (somatotrophin) et al. 1997.

benthamiana 1992 1993 (Firek et al... tuberosum Glycine max P. 1996) (Bruyns et al.. 1999) (Vaquero et al.. 1997) (Phillips et al. tabacum 1996 Phosphonate ester NP hapten Substance P (neuropeptide) Phytochrome Human creatine kinase Phytochrome AMCV Fungal cutinase Streptococcus mutans adhesin Streptococcus mutans adhesin TMV Cutinase RKN secretion BNYVV Human creatine kinase Human creatine kinase β-1. apoplast Apoplast. 1993) (van Engelen et al.. Recombinant antibodies expressed in transgenic plants Year Antibody format Antigen Plant organ Cellular location Transformed species Reference 1989 1990 1991 IgG1 IgM VH domain Leaf Leaf Leaf (Hiatt et al. benthamiana N. 1997) (Francisco et al.. 1998) (Torres et al. BNYVV – beet nectrotic yellow vein virus. scFv-IT – scFv-bryodin-immunotoxin. benthamiana N. tabacum N.and extra-cellular Cytosol Nucleolus N. sativa T. 1997) (Fiedler et al. benthamiana (Khoudi et al. 1998) Leaf ER Secretory pathway Cytosol S. 1993) 1993 1993 1994 1994 scFv IgG1 Fab scFv scFv IgG IgG1 ER ER chloroplast Intra.. tabacum N. 1996) (Baum et al.. tabacum N. 1989) (Düring et al... tabacum (Ma et al. apoplast 1999 scFv Leaf Apoplast 2000 2000 scFv scFv 38C13 mouse B cell lymphoma CEA TMV Plant Leaf ER. tabacum N. 1997) Oxazolone HSV-2 tuber Plant (Artsaenko et al. apoplast Medicago sativa N. tabacum N.. 1992) (De Neve et al..284 Table 3. 1993a) (Tavladoraki et al. 1999)F CEA – carcinoembryonic antigen... aestivum N. 1999d) (McCormick et al. tabacum (Stöger et al.. tabacum N. . tabacum tissue culture S. 1994) (Ma et al. tabacum N. sativa Suspension cells N... in press) 1997 1997 1997 1997 1997 1998 1998 1998 scFv Humanized IgG1 scFv 1999 1999 1999 1999 IgG scFv scFv bi-scFv Dihydro-flavonol 4-reductase Human IgG CEA Tospoviruses TMV 1999 1999 scFv scFv TMV CEA Plant Cell Cytosol ER. thaliana (De Wilde et al. apoplast ER.. tabacum N. benthamiana N. thaliana N. tabacum O. 1995) (Schouten et al. HSV-2 – herpes simplex virus-2.... 1996) (Fecker et al.. tabacum Suspension cells N. 1999) (Fischer et al. 1991) Leaf Leaf Leaf Leaf Root Leaf Apoplast Cytosol Apoplast Apoplast N. tabacum A. 1995) 1995 1996 1996 1996 1996 IgG scFv IgM scFv scFv Leaf Leaf Leaf root Leaf Leaf (Voss et al. 2000) (Schillberg et al. tabacum N.. tabacum N. 1990) (Benvenuto et al.4-endoglucanase Oxazolone Abscisic acid Abscisic acid CD-40 A. ER – endoplasmic reticulum.. 1996) Root Leaf Leaf Seed Plant Cytosol ER ER ER Apoplast (Schouten et al. 1994) 1995 IgA/G Leaf Apoplast N.... 1998) (Zeitlin et al.. tabacum N. tabacum N. RKN – root knot nematode. AMCV – Artichoke mottle crinkle virus. 1999) O. tuberosum N. hybrida (De Jaeger et al.... membrane (Zimmermann et al. TMV – tobacco mosaic virus. 1997) (Fiedler et al. 1999) (Franconi et al. 1998) Plant Leaf Plant Leaf Apoplast Transient expression ER. 1996) IgG1 Fab scFv scFv scFv scFv scFv-IT Leaf Apoplast ER Apoplast Apoplast Cytoplasm ER Apoplast N.. tabacum (Owen et al..

tuberosum Virus particle Virus particle A... scFvs and other recombinant proteins (McCormick et al. S. tabacum Virus particle L. tabacum. Lactuca sativa N.. 1996) have attracted interest because high yields of protein can be produced rapidly. 1996). tuberosum Virus particle (Mason et al.... 1999. tuberosum S. 1997) (Dalsgaard et al. Particle bombardment usually reaches only a few cells and for transcription the DNA has to reach the cell nucleus (Christou. flexible and straightforward and often use either agrobacterial or viral vectors. CA. S.. tabacum N... 1998) (Modelska et al. In contrast. tabacum. Viral vectors (Scholthof et al. 1997) (Yusibov et al. transient gene expression systems are rapid. by comparison to stable plant transformation. 1999) Lupinus luteus. or infection with modified viral vectors. Commercial field trials are proceeding for the production of recombinant proteins using viral vectors. 1997) (Carrillo et al. For example.. 1996). tabacum. This makes transient expression suitable for verifying that the gene product is functional before moving on to large-scale production in transgenic plants (Kapila et al. tuberosum S. The overall level of transformation varies between these three systems. thaliana (Mason et al. tuberosum (Mason et al... which transiently generates many transcripts of the gene of interest.. 1998) (Tacket et al.. esculentum N.. 1997) (Ehsani et al. carota (Kapusta et al. 1999) 1996 1997 1997 1997 1997 1998 1998 1998 1998 1998 1998 1999 1999 1999 1999 genic plants. 1998) S.. There are three major transient expression systems to deliver a gene to plant cells: delivery of projectiles coated with ‘naked DNA’ by particle bombardment... Stable plant transformation requires a considerable investment in time before the expressed proteins can be analysed. 1999) (Porceddu et al. 1998) (Arakawa et al.. S. 1996).. Agroinfiltration targets many more cells than particle bombardment and the T-DNA harboring the gene of interest is actively transferred into the nucleus with the aid of several bacterial proteins (Kapila et al. 1998a) (Arakawa et al. 1995) (Porta et al. Kumagai et al. tuberosum Virus particle S. 1996) N. tuberosum S. . 1995) (McGarvey et al. 1995) (Haq et al. Recombinant vaccines expressed in plants Year Vaccine antigen Transformed species Reference 1992 1995 1995 1995 1996 Hepatitis virus B surface antigen Malaria parasite antigen Rabies virus glycoprotein Escherichia coli heat-labile enterotoxin Human rhinovirus 14 (HRV-14) and human immunodeficiency virus type (HIV-1) epitopes Norwalk virus capsid protein Diabetes-associated autoantigen Hepatitis B surface proteins Mink Enteritis Virus epitope Rabies and HIV epitopes Foot and mouth disease virus VP1 structural protein Escherichia coli heat-labile enterotoxin Escherichia coli heat-labile enterotoxin Rabies virus Cholera toxin B subunit Human insulin-Cholera toxin B subunit fusion protein Foot and mouth disease virus VP1 structural protein Hepatitis B virus surface antigen Human cytomegalovirus glycoprotein B Diabetes-associated autoantigen N.. Transient expression is rapid and results on protein expression can be obtained in days (Kapila et al.. tabacum. 2000).. the Large Scale Biology Corporation (formerly BioSource Technologies. A viral vector can systemically infect most cells in a plant and transcription of the introduced gene in RNA viruses is achieved by viral replication in the cytoplasm.. 1996) (Ma et al. USA) is using a tobacco mosaic virus-based vector for production of Hepatitis B surface antigen.. 1996).. Vacaville. infiltration of intact tissue with recombinant agrobacteria (agroinfiltration). 1992) (Turpen et al. 1998b) Medicago sativa (Wigdorovitz et al. tuberosum N.285 Table 4. D. 1999) (Tackaberry et al.

Using agroinfiltration. 1999). the mouse–human chimeric heavy and light chain genes were integrated into two vectors in two separate recombinant Agrobacterium strains and these two strains were simultaneously infiltrated into leaves. In agroinfiltration. 1996).. Thus.expression with a bacterial vector In agroinfiltration. agroinfiltration can provide milligram amounts of a recombinant protein within a week (Vaquero et al. where it is transcribed and this leads to transient expression of the gene of interest (Kapila et al. Thus. Improvement of this technique may involve the increase of inoculation efficiency by combining the cloned recombinant viral DNAs with particle bombardment or Agrobacterium . are easily transmissible by mechanical inoculation and can spread from plant to plant. Kapila et al. the gene of interest is cloned into the genome of a viral plant pathogen under the control of a strong subgenomic promoter. The coat protein is essential for long distance. Hendy et al.. agrobacteria carrying the expression vector are delivered into leaf tissue by vacuum infiltration. This is an important issue because it dramatically accelerates the development of plant lines producing recombinant therapeutics.. A major advantage of this technique is that multiple genes present in different populations of agrobacteria can be simultaneously expressed. 1996) and no selection method to identify transformed cells is required since the leaf tissue is only used for transient protein production. For full size chimeric antibody expression. the assembly of complex multimeric proteins can be tested in planta (Vaquero et al. 1996). This approach has been adapted to express the heavy and light chain of a full size antibody from two different viral vectors (Verch et al. systemic viral infection and the scFv was expressed throughout the entire plant. this can only be achieved by time consuming crossing experiments with individual transgenic plant lines. A bacterial suspension is then used for vacuum infiltration of leaves (An. unpublished results). Some plant viruses have a wide host range. Here. it is unsuitable for the expression of larger amounts of foreign proteins. genes of interest are cloned into binary vectors that are transferred into suitable Agrobacterium strains.. Here. This promoter is duplicated and drives the transcription of the viral coat protein gene. As in conventional methods for generating transgenic plants. agroinfiltration is rapid and yields sufficient quantities of protein for initial characterization of protein stability and protein function. 1996).286 Although particle bombardment can be used to test recombinant protein stability before stable transformation. Typically. 1996) share several advantages with agroinfiltration. 1999. 1999). It is more important for the regeneration of transgenic cereal crops (Christou... 1998). 1985. McCormick et al. which permits the use of smaller plasmid vectors... Fischer & C. making it possible to rapidly infect large numbers of plants with recombinant viruses. diabodies and multi-component protein complexes (R. The scFv coding region was inserted into the viral genome under the control of the strong subgenomic coat protein promoter.. Infectious recombinant viral transcripts are used to infect plants and produce the target protein. but is generally limited to proteins smaller than 30 kDa. Viral vectors have been used to express single chain antibodies in plants (Franconi et al. Agrobacterial proteins then catalyze the transfer of the gene of interest into the host cells and protein expression can be detected three days after infiltration. 1999). we transiently expressed scFvs. For transgenic plants. We transiently expressed an scFv in plants. individual heavy and light chains as well as full size mouse–human chimeric anti-carcinoembryonic antigen (CEA) antibodies in plant leaves (Vaquero et al. This technique is also suitable for the expression of scFv-fusion proteins. the transferred T-DNA does not integrate into the host chromosome but is present in the nucleus. Functional full size chimeric antibodies were assembled in vivo by simultaneous expression of both genes in the host cells. Vaquero. Agroinfiltration... Importantly. Recombinant viral vectors Viral vectors (Scholthof et al. agroinfiltration can be scaled up to produce tens of milligrams of recombinant protein and may even prove suitable for pre-clinical trials without the need for production of stably transformed plants. 1999). 1999). 1999. Target genes are expressed at high levels because of the high level of multiplication during virus replication (Porta & Lomonossoff. we concentrate on agroinfiltration as a method to test antibody production before producing stably transformed plants. using a tobacco mosaic virus (TMV) based vector (Verch et al. each expressing a single component of the multimer.

and a eubacterial glucanase has been reported to reach 26% TSP in the mouse eared cress. Initial problems can be identified and eliminated so that the likelihood of regenerating the desired transgenic line is significantly improved. 1984. rice can be transformed by Agrobacterium (Chan et al. 1993). High intensity agriculture can produce suprisingly large amounts of biomass. which prevents dental caries .. 1983.. 1997) and methods have been developed for transforming other monocots. 2000). such as tobacco and pea (Horsch et al.9 9. The quantity of recombinant protein that can be harvested is only limited by the number of hectares that can be planted with transgenics. 1997). apoplast targeted recombinant phytase accumulates to ≈14% total soluble protein (TSP) in tobacco leaves (Verwoerd et al.9 33.. we summarize the yield per hectare of several crop species as a guide to what levels of biomass can be produced. Some recombinant proteins already reach very high expression levels. Assuming that the levels of production seen on the laboratory scale could be at least kept constant in the field..6 24. Plants are the premier heterologous system for the production of secretory IgA antibodies (Ma et al.. Yields of several plant species in tonnes per hectare Crop Crop yield (tonnes/hectare)∗ Reference Banana Cabbage Eggplant Lettuce Maize Peanut Peas Potato Rapeseed Rice Tobacco Tobacco 16. Table 5. The generation of transgenic plants uses two principle technologies: Agrobacterium mediated gene transfer to dicots.1 124. for example...5 6. Cramer et al. stable transgenic plants are the most attractive strategy. we compare plants to the classical expression systems and show the advantages of plants. However. However.6 2. transient expression is a prerequisite to stable transformation because it allows both the expression vectors and protein stability to be tested. Sheen.1 8. Importantly. Planet Biotechnology (Mountain View.287 based techniques. 1995). such as wheat and corn (Christou. In Table 1.4 2. Arabidopsis thaliana (Ziegler et al. 1996). (Ma et al. Clearly. 1985). 1983) FAO FAO Stable plant transformation Stable transformation is defined by the integration of a target gene into the host plant genome. Agrobacterium has a restricted host range and does not efficiently infect monocots but is the most widely used technique for dicot transformation. Transformation is followed by selection for cells with stably integrated copies of the target gene by following a selectable resistance gene that is introduced in the expression vector.7 FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO (Long.. Taken from the Food and Agriculture Organization’s on line database (http://apps.4 2. and that for every 170 tonnes harvested. or biolistic delivery of genes to monocots.2∗ 170–200∗∗ Tomato Wheat 59. average expression levels of recombinant antibodies in stably transformed plants are usually on the level of 0.8% TSP (Fiedler et al. In Table 5. 1998) but have reached 6. 1996).5–2% TSP (Conrad & Fiedler. coli and Agrobacterium. for example intensive cultivation of tobacco plants can produce approximately 170 metric tonnes of biomass per hectare (Sheen. Hiei et al..∗ : smoking tobacco.fao.org/).3 26. 100 tonnes are harvested leaves: a single hectare could yield 50 kg of secretory IgA All yields refer to production in developed North America in 1999. CA) are concentrating on using plants to produce the Streptococcus mutans specific Guy’s-13 antibody. 1995). like agroinfection (Shen & Hohn. 1995) or 100 kg of recombinant glucocerebrosidase (Cramer et al. ∗∗ : close cropping and mowing.. the gene of interest is cloned into a binary vector that can be moved between E.. plants have some unique features not found in bacterial or mammalian systems. 1994. For transforming plants. Hiei et al. 1993. Stable transformation of plants depends on the plant variety and it can take 3–9 months for plants to be available for testing the expressed protein. The transformed Agrobacterium itself delivers the target gene into the host cell genome. Expression in stably transformed plants When long term production of a recombinant antibody is necessary. particularly their safety and low cost. 1995).5 1.

1999). have reached levels of up to 1. the glycosylation of proteins intended for in vivo administration should be analysed in detail (Bardor et al. 1999). Staub et al. require no special maintenance and have a long shelf life (Conrad et al. 1993.0% of total soluble protein (De Jaeger et al. Zimmermann et al. Schillberg et al.. De Wilde et al.. 1994. 1992..288 (Ma et al. which is inherently biologically contained..3% of the total soluble protein (TSP) in tobacco leaves (Hiatt et al. Additionally. Active human somatotrophin was expressed in transgenic plastids in tobacco and reached 7% of the total soluble protein (R. There is a report where cytosolic scFvs. 1997) and levels of secretory IgA up to 500 µg per gram leaf material (Ma et al.. 1998). 1998). Tavladoraki et al. In the majority of transgenic plants expressing cytosolic scFvs. Schouten et al. Important considerations for pharmaceutical expression in plants In this section.... 1998). Therefore. Voss et al.. 1995. 1996. 1993b. 1996. The plant pattern of protein glycosylation and Nlinked glycan processing differs from that of animals. The low costs of producing recombinant antibodies in plants are as great a benefit as the increased safety and authentic post-translational modification pathways.. .... Overall expression levels of different antibodies in stably transformed plants vary.. Ma et al.. 1998). Baum et al. Bassuner. 1995. 1998). The high cytosolic expression level may be because the in vitro antibody selection used in phage display naturally selects more stable antibody fragments. However. presumably because scFv fragments require only minor post-translational processing. we discuss the technical considerations that are important for high level pharmaceutical protein expression. 1996. 1998)..35% (van Engelen et al. such as sIgA. 1990. Significant increases in recombinant antibody yield have been observed when antibodies are targeted to the secretory pathway instead of the cytosol (Conrad & Fiedler. Fecker et al. Ma et al. levels were found to be very low or at the detection limit (Owen et al. This is not an upper limit. isolated from a phage display library. Russell and colleagues at the Integrated Protein Technologies unit of Monsanto have reported that transformed chloroplasts can be used as a high capacity production system. which are extremely stable. 1995). Schouten et al. chloroplasts and endoplasmic reticulum (ER) (Düring et al.. 2000). 1996. 1996. It will be interesting to determine if chloroplasts will be capable of synthesizing complex eukaryotic proteins that require post-translational modifications or subunit assembly. Similar approaches are now underway to produce other antibodies under a collaboration between EPIcyte pharmaceuticals and ProdiGene. coli and that these savings will be greater as production reaches agricultural cropping scales or as methods are developed to increase expression levels. This has the benefit that both the product and the production system itself can be stored almost indefinitely. 1994) to 1. Artsaenko et al. because transgenic plants have been identified with expression levels of scFvs in leaves reaching 6. 1995. personal communication.. may be useful for the expression of other pharmaceutical proteins. Firek et al.. but this is still an exception. Sub-cellular protein targeting Expression levels of recombinant antibodies in plants can be enhanced by exploiting the innate protein sorting and targeting mechanisms that plant cells use to target host proteins to organelles. which range from transcriptional modifications to changes in the sub-cellular destination of the newly synthesized recombinant protein. This justifies the use of plants as an inexpensive source for producing recombinant proteins that eliminates the disadvantages associated with microbial or animal cell systems. Targeting proteins for secretion to the intercellular space beneath the cell wall (apoplast) has advantages for downstream processing and also leads to significant levels of expression but ER retention can give 10–100-fold higher yields (Conrad & Fiedler.. It has been estimated that plant-expressed proteins are 10–50-fold less expensive than those made in E.. 1996. In a recent report. it is unclear to what extent the chloroplast protein synthesis pathway can accommodate eukaryotic proteins. with expression of full size IgG under the control of the 35S promoter ranging from 0. Schouten et al. 1998). plant genetic material is readily stored in seeds or tubers.. This a remarkable result and indicates that chloroplast based expression... and in some cases recombinant proteins could be immunogenic through their glycosylation.8% of the TSP (Fiedler et al. Intracellular expression of rAbs in the cytoplasm has only been achieved using scFv fragments (Owen et al. 1992. 1989). Conrad & Fiedler. Recombinant antibodies have been targeted to the following compartments of plant cells: the intercellular space...

289 Protein storage in tissues. rice. Seeds are protein rich storage organs that can be stored almost indefinitely and can be exploited as storage containers for recombinant proteins (Fiedler and Conrad. a prerequisite was that expressed proteins could be stored in harvested tissues. distribution and processing. 2000). and edible vaccines (Walmsley & Arntzen. Pharmaceutically valuable proteins produced in plants As medical and biological knowledge of many diseases increases. 1998. Therefore. corn.. 1999). to target the protein for seed specific expression. Using this strategy. plasma proteins and diagnostic reagents are targets for expression in plants because their conventional production or purification is often expensive and can bear risk of pathogen contamination. legumes) may be used because they have a lower content of toxic compounds than model species. As described earlier. HSA can be safely made in plants as can interleukins.. if long-term storage is required. We foresee crop based expression systems (wheat. for example human serum albumin (HSA) is a non-glycosylated protein with a worldwide demand approaching 550 tonnes of purified protein a year. 1995. human enzymes and recombinant vaccines (Tables 2–4). Therapeutic antibody production in plants Antibodies are an instructive example of the expression of pharmaceutically valuable proteins in plants (Hiatt. 1998) and screens to identify stable cytosolic antibody scaffolds (Worn et al. like tobacco. Transformation of major crop plants is now becoming more straightforward.. The worldwide demand for some of these proteins is large. and there is an existing infrastructure for crop cultivation. isolated HSA does bear some risks of viral contamination and there is a market for sources of safer HSA. Plants are likely to feature highly as an alternative to using transgenic animals to produce these proteins. it is isolated from human blood donations and is therefore relatively inexpensive. 1997). 1997). Potato tubers have also been used as storage containers with expression levels reaching 2% TSP and cold storage for 18 months resulting in only a 50% loss of functional antibody (Artsaenko et al. 1998). there were no losses in scFv specificity or antigen binding activity (Fiedler et al. Hiatt & Ma. 1993). This is likely to be important in the future. Interestingly. a key breakthrough in making molecular farming in plants a reality was the demonstration of functional antibody expression in .. Optimization at the level of the gene The pattern of codon usage in plants differs to that of animals... The number of mammalian proteins expressed in plants is expanding and include antibodies. many novel proteins that could be used for treatment have been identified. ER retention gave increases in scFv accumulation and scFvs can be stored for up to a year in seed at room temperature without losses. Hiatt. 1998).. adroit selection of genes to suppress by anti sense expression may be a realistic method to dramatically increase the yield of co-introduced pharmaceutical protein genes in seed. 1990. Conrad and Fiedler. When leaves from transgenic tobacco plants expressing ERretained scFvs were dried and stored for more than three weeks. 1992. arcelin levels were enhanced and reached 24% of the total seed protein (Goossens et al. but here we focus on the more advanced fields of antibody expression and vaccine production. This has widely been shown to be the case and emphasizes the versatility of plants as an expression system. Conventionally. harvesting. Hiatt et al. through the sequencing of the human genome and medical research. plasma proteins. seeds and tubers For plants to fulfill their potential as a production system. Further improvements in expression may come from using tissue specific promoters. Another application of molecular farming in plants is the production of vaccine antigens. tubers and seeds. the production of a bean arcelin in Arabidopsis thaliana seeds was increased by the expression of an antisense gene for the seed storage protein 2S albumin. These include recombinant antibodies and it is clear this will expand to include more proteins in the future. Recombinant antibodies. interferons and human enzymes (Table 2). Conrad et al. translational enhancement with viral sequences (Gallie. 1991. improvement of transcript stability. 2000). The most promising approach for protein expression and in field production is to target the protein to the ER and. As with expression in leaves. but modifying the composition of the heterologous cDNA to meet the plant pattern can increase the rate of translation (Kusnadi et al. However.

Tavladoraki et al. It was first expressed in tobacco by sequentially crossing plants expressing its individual polypeptide components. In vivo assembly of full-size cT84. 1999). 1996. 1999d). 1996).. SIgA is a complex multi-subunit antibody with great potential for use in topical immunotherapy. These formats include full-size antibodies (Ma et al. since single plant cells are capable of expressing recombinant secretory IgA (sIgA) (Ma et al. A recombinant single-chain Fv antibody (scFvT84. 1995). such as phage display. 1993b. 1975) and recombinant antibodies are essential therapeutic and diagnostic tools used in medicine.. a wide range of different recombinant antibody formats have been successfully expressed in many plant species (Table 3). 1995. 1990). antibodies are likely to only become more important in the future and the demand for an economical. 1994). Fab fragments (De Neve et al. Fecker et al.. single chain antibody fragments (scFvs) (Owen et al. Thus. smaller antibody fragments or antibody-fusion proteins linked to enzymes. efficient expression system is likely to grow. derived from the parental murine mAbT84.. 1993). Voss et al. the same group showed that recombinant sIgA specific for adhesion proteins from the oral pathogen Streptococcus mutans could prevent oral streptococcal colonization in human volunteers for up to four months (Ma et al. 1995.. 1989) and 1990 (Düring et al. The rapid development of combinatorial approaches. Three years later. Therefore.. Secretory IgA Plants have a great advantage over animal cell expression systems. were engineered into a plant expression vector. 1993.. bi-specific scFv fragments (Fischer et al. 1990). Modern recombinant DNA techniques and antibody engineering have broadened the range of applications for recombinant antibodies. 1999). The circle will be closed when the first plant expressed antibodies are approved by the regulatory authorities and come onto the market as diagnostic or therapeutic products. molecular farming has come virtually full circle. in press) and chimeric antibodies (Vaquero et al. 1995. Firek et al.. This has only recently become possible in single animal cells (Chintalacharuvu & Morrison... a joining chain and the secretory component. Chimeric T84. After the demonstration that functional full size rAbs could be expressed in transgenic plants in 1989 (Hiatt et al.... membrane anchored scFv (Schillberg et al. Artsaenko et al. allow the isolation of rAbs recognizing almost any target antigen and the fine-tuning of these rAbs toward desired properties (Winter et al. These advances have made possible the production of novel polypeptides with desirable properties (Figure 1). Bookman.66 heavy and light chain genes were constructed by exchanging the mouse light and heavy chain constant domain sequences with their human counterparts and cloned into two independent plant expression vectors. It is the major antibody found in mucosal secretions and is made of two immunoglobulin chains.... 1997.. Gerstmayer et al. We have shown that it is possible to transiently express tumour (carcinoembryonic antigen) specific single chain and chimeric full size antibodies in tobacco leaves. 1997). Upscaling the transient system permitted purification of significant (milligram) amounts of functional recombinant antibodies from tobacco leaf extracts within a week (Vaquero et al. 1989. 1993.. 1989) to the production of complex antibodies in plants and their use as pharmaceutical reagents. biological response modifiers or toxins ( Shin et al. 1992.66 specific for the human carcinoembryonic antigen. Vaccines from plants Vaccination has been one of the greatest advances in medical science and has dramatically improved human life expectancy and quality of health. For example.. from proof of principle with the expression of model antibodies in 1989 (Hiatt et al. 1998).290 tobacco leaves (Hiatt et al. Baum et al.66). The importance of this is underscored by the fact that monoclonal antibodies (Koehler & Milstein. 1991). the life sciences and biotechnology (Winter & Milstein. 1994... 1996).. for antibodies at least..66 was achieved by simultaneous expression of the light and heavy chains after vacuum infiltration of tobacco leaves with two populations of recombinant Agrobacterium. Fiedler & Conrad. 1998). Düring et al.66) and a full-size mouse/human chimeric antibody (cT84.. 1995) and proved that plants could be used as a production system for sIgA suitable for use in passive immunotherapy. Vaccines are the most cost effective form of health care . human and animal health care. Schouten et al. This permitted the production of high levels of recombinant sIgA (500 µg/gram) (Ma et al... De Wilde et al. We anticipate that plant produced sIgA will become widely used in the future for the generation of passive immunity because of their stability in the mucosa. 1996.

. A number of plant species has been used for the generation and propagation of cell suspension cultures.. 1988)... Whitelam et al. Many edible plants have been genetically transformed and tomatoes and bananas are good candidates for edible vaccine production for humans while cereals may be more suitable for animal immunisation. 1999). alfalfa (Daniell & Edwards. Walmsley & Arntzen. an effective edible hepatitis B vaccine has been generated using transgenic lupin and lettuce plants expressing the hepatitis B surface antigen..int/gpv/) but their world wide distribution is hampered. 1997). 1992.. Tobacco suspension cells have been used for the production of an scFv-fusion protein with a ribosome inactivating protein (Bryodin) with yields of 30 mg/l (Francisco et al. 1999). while rhizosecretion is the release of proteins from transgenic plant roots into a surrounding hydroponic medium (Borisjuk et al. distribution and processing..and T-cell mediated immune responses using plants as a source of ‘edible vaccines’. 1989).. Suspension cells can be used to produce and secrete proteins under carefully controlled certified conditions. 1996). 1995). antibody fragments and fusion proteins in transgenic plant cell suspension systems including Nicotiana tabacum cv. 1995) and tobacco (Nagata et al. Alternative plant expression systems Intact tobacco plants are not the only expression system available for plant based molecular farming.. When clinical use of recombinant proteins is intended. 2000). As discussed earlier... Hood et al. 1994). It was proposed to induce B. Taxus (Seki et al. Cereals have advantages for antibody production over ‘model’ species such as tobacco because they have a lower content of toxic secondary metabolites and there is a well-organised infrastructure for their production.. we have expressed full-size antibodies.. effective methods have to be developed for the biological containment of vaccine traits. Such vaccines would not require cold storage or sophisticated expertise for their distribution and use throughout the developing countries. 1989. foot and mouth disease (Wigdorovitz et al. For edible vaccines to become widely used and useful. 1991.291 (World Health Organisation: www. Mice fed raw potato produced a serum and mucosal immune response to the antigen. Edible vaccines will need an infrastructure for their distribution and administration to the public to ensure they are as effective as current vaccines. Nicotiana tabacum BY-2 cells (Nagata .who. 1997).. there have to be internal controls for the level of vaccine expression in every plant and their stability and efficacy need to be improved. 1999). We focus on using suspension cells for the production of recombinant proteins and antibodies in plants and plant cell cultures. several issues have to be considered. 1992). 1995).. Fischer et al. to important monocot or dicot crop plants like rice (Chen et al. Nagata et al. Therefore. 1994). 1998a).. Plant suspension cells are a model system that can be easily transformed and cultivated on a very large scale in fermenters (Fischer et al. their production under defined. but rhizosecretion has yet to be broadly used (Hooker et al. the expression of recombinant antibodies and antibody fragments in plants is well established (Hiatt et al. ranging from model systems like Arabidopsis (Desikan et al.. especially in developing countries. Further. 1998b) and cholera (Arakawa et al. 1999b). plant suspension cells and by ‘rhizosecretion’ from engineered plant roots. Furthermore. 1995. soybean (Hoehl et al. There are options for the production of proteins in seeds... 1999). Further work has gone on to show that edible vaccines may be feasible for a range of antigens. 1999. Mice and humans fed transgenic plant material produced hepatitis B specific antibodies (Kapusta et al.. Bisaria & Panda. 1999b). where the vaccine antigens are eaten in a fruit or raw vegetable (Mason & Arntzen. Petite Havana SR-1 (Voss et al. Compared to the classical expression systems the number of applications is still relatively small (Kieran et al. Ma & Vine. When recombinant proteins are expressed in cereal seed.. 1997). Importantly. Plant suspension cells can be grown in shake flasks or fermenters to produce recombinant proteins after transformation (Fischer et al. coli heat labile enterotoxin B subunit (Haq et al.. including rabies virus (McGarvey et al. 1996) to Catharanthus (Van Der Heijden et al... such as for expression of avidin in corn. 1999b). Norwalk virus (Mason et al.. 1995). 1997. The principle of edible vaccine activity was proven for transgenic potatoes producing the enterotoxigenic E. autoimmune diabetes (Arakawa et al.. 1990. Plants can produce a range of immunogenic antigens (Table 4). controllable and sterile conditions with straightforward purification protocols may be advantageous. the protein can be collected and extracted directly from the kernels (Hood et al.

This approach is not applicable for the downstream processing of IgM antibodies. Although there are established protocols available for purification of antibodies produced by animal or microbial sources. Downstream processing of full-length IgG antibodies is relatively straightforward because Protein-A and Protein-G are useful ligands for affinity chromatography. we could obtain expression levels between 2–20 µg of recombinant antibody per gram fresh cell weight. 1990. Mariani & Tarditi.. high capacity and that are inexpensive will need to be developed. or most recombinant antibodies including scFvs and scFv fusion proteins. vacuole. The cytosol is generally unsuitable as a recombinant protein storage compartment. 1985). 1997). our preferred method is to target proteins for secretion and capture them from the culture supernatant or release them from the cell by mild enzymatic cell wall digestion (Fischer et al. 1999c).292 et al. 1997). Recombinant proteins expressed in plant cell suspension cultures are found in the culture supernatant or retained within the cells. chloroplast. This shows that antibody purification from plants is essentially straightforward with no complications that could prevent the use of plants as an expression system. This level could be significantly increased by targeting of the protein to the ER as well as by optimizing cultivation condi- tions with controlled amino-acid supplementation or elicitation. 1992. there is little available data on the purification of recombinant antibodies from plants. Our data demonstrate that full-size antibodies can be purified from plant cell extracts on protein-A and protein-G based affinity matrices in a similar manner to antibodies purified from animal sources. 1992).. pea. Moloney & Holbrook. Using our standard plant expression vector. 1999c). intracellular membranes) (Moloney & Holbrook. Thus. 1997. Miele. wheat and rice (Torres et al. 1997. Antibodies secreted to the intercellular space of plant cells were released by partial enzymatic lysis of the cell wall and this was the superior method for isolation of functional antibodies. since it causes release of phenolic substances or proteases that reduce protein yield. Gel filtration served as a polishing step for the removal of rAb-dimers and for exchange of the rAbs into a suitable storage buffer. For most recombinant proteins. particle bombardment (Christou. We established a purification protocol for full-size antibodies produced in plant cell suspension cultures (Fischer et al. Recombinant proteins smaller than 20–30 kDa can pass through the plant cell wall and are secreted into the culture medium but larger proteins tend to be retained in the apoplast. 1979). 1997).. the latest developments in downstream processing such as perfusion chromatography and expanded bed tech- . novel strategies that have a high processing speed. 1999). 1996). Affinity chromatography using a Protein-A matrix as the first step efficiently removed contaminant plant proteins and gave a 100-fold concentration of the recombinant protein. 1993). 1987) or viral vectors (Porta & Lomonossoff. Purification strategies for proteins expressed in plants Highly efficient purification schemes are a prerequisite for the use of recombinant proteins as pharmaceuticals (Baker & Harkonen.. Koncz & Schell. more than 80% of expressed full size IgG can be recovered from suspension cultured plant cells (Fischer et al. Using this method. plant suspension culture cells. electroporation of protoplasts (Lindsey & Jones. 1998) or to intracellular organelles (ER. To make large scale bioprocessing more efficient. This has the advantage that transient expression of the foreign gene can be detected 2–3 days after cocultivation. protect them from proteolytic degradation and to increase accumulation levels (Kusnadi et al. The targeting signals can be used to retain recombinant proteins within distinct compartments of the cells to preserve integrity.. This localisation is dependent on the presence of targeting/leader peptides (of plant or heterologous origin) in the recombinant protein and on the permeability of the plant cell wall to macromolecules (Carpita et al. which has several drawbacks.... 1997) and are an important consideration in designing molecular farming systems. Tobacco suspension cell lines for recombinant antibody production Transfer of a foreign gene into plant suspension cells can be performed using Agrobacterium-mediated transformation (Horsch et al. Recombinant targeting signals can be used to direct the protein for secretion (Magnuson et al.. 1986). leaves or seeds (Moloney & Holbrook. Intra-cellular protein retention makes the disruption of the cells necessary prior to protein purification. 1985. 1999c). The BY-2 cell line can be directly transformed by co-cultivation of suspension cells and Agrobacterium (An. Murano.

000 hectares of land (assuming an expression level of 1% TSP in tobacco). silica) for the development of a specific affinity matrix for a given recombinant protein (Murray et al. Engineered affinity tags may enable improved handling of large clarified sample volumes. was engineered in fusion to a barley α-amylase signal sequence... This illustrates how a relatively abundant protein with a rich natural source can still be produced less expensively in plants. The maize avidin is functional and now commercially available (Sigma-Aldrich product # A8706). 1999)... As mentioned earlier.293 nology in combination with tangential flow filtration should be applied. β-Glucuronidase production in plants is also commercial (Witcher et al. Transgenic animals are limited by the time needed to raise a herd of animals producing the recombinant protein. The demand for human serum albumin is in the range of 550 metric tons per year and it may take years to establish a herd of transgenic animals producing enough protein to meet this demand. The combination of the latest developments in downstream processing and affinity chromatography may lead to significant advances in the large scale production of inexpensive diagnostic and therapeutic proteins by molecular farming. An interesting case study for the farming of a recombinant protein was reported by Hood and co-workers for the production of recombinant avidin (Hood et al. such as their production in mammalian tissue culture. It has been estimated that the worldwide demand for human serum albumin could be met by transgenic cultivation on 30. transgenic plants can be rapidly scaled up to field scale cultivation. targeting the avidin to the cytosol was completely toxic to engineered maize.. the savings from expressing antibodies in plants will be even higher.. The rationale was to produce avidin in transgenic corn and determine if this could compete with egg white as a commercial source of the protein. Due to potential immunogenicity of certain tags (FLAG.. glass. Zwick et al. The demand for many pharmaceutical proteins is large. (Mountain View.. 1999). we need to distinguish between the generation of N. Clinical trials of plant produced pharmaceuticals The first clinical trial of plant-based immunotherapy was reported by Planet Biotechnology. This will overcome the diffusional limitations experienced with most chromatographic resins (Fahrner et al. 1998) and the costs of producing aprotinin in plants are comparable with extracting it from its natural source. 1999. GST) it may be particularly important to use specific. Such peptides can be identified by epitope mapping using pepscan or phage peptide display technologies for the identification of linear peptide sequences. 1997). The authors estimated that plant produced avidin is 10-fold less expensive than avidin extracted from eggs. MBP. In contrast. 1998). Kusnadi et al. which is less than one thousandth of the total cultivated soil in the USA (32 million hectares). 1998. 1998)... to allow controlled capture and release during processing and multiple uses of a synthetic affinity matrix. The peptides are immobilized on a solid support. Modifications of the phage peptide display technology also permit the identification of mimotopes. Plants that expressed high levels of avidin in the secretory pathway were either partially or completely male sterile. Considering that there are no natural sources of recombinant antibodies as inexpensive as using chicken eggs as a source of avidin. 1997. codon optimised for the preferred maize codon usage pattern. This targeted the recombinant protein to the secretory pathway and targeting was a crucial factor. Commercial aspects of molecular farming The commercial interest in molecular farming is that it can produce recombinant proteins at a lower cost than alternatives. and any transgenic production system has to be capable of meeting the demand. synthetic peptide affinity ligands for the purification of therapeutic proteins. 1997) and the development of stable synthetic peptides that reversibly bind the recombinant protein of interest. A chicken avidin cDNA. Hood et al. either in a linear or a Cys-Cys-constrained library (McConnell et al. Avidin is widely used as a diagnostic reagent and is a relatively abundant eukaryotic protein found in egg white. bovine lung (Zhong et al. minimise processing time and avoid proteolytic and oxidative degradation of recombinant proteins. kilogram quantities of a recombinant protein can be obtained from as little as a hectare of transgenic tobacco. The novel drug CaroRxTM is based .. from which it is routinely purified. Inc. Avidin could be reproducibly produced at 230 mg per kg of maize seed (Hood et al. 1997). CA). Here. The synthetic versions of identified binding peptides can be immobilized on an activated matrix (Sepharose. In contrast.or Cterminal gene fusions (tags) for affinity purification (Nilsson et al.

If a laboratory develops its own proprietary transformation and expression system.. similar to mice immunized with the native 38C13 IgM-keyhole limpet hemocyanin conjugate vaccine (McCormick et al. Monsanto Integrated Protein Technologies unit. This rapid production system for generating tumor-specific protein vaccines may provide a viable strategy for the treatment of non-Hodgkin’s lymphoma. The company is also cultivating transgenic soybeans that produce humanized antibodies against herpes simplex virus 2 (HSV-2). stronger promoters in intact plants. This is highly interesting because through this technology. 1998. Large-scale fermentation may be a practical method for producing recombinant pharmaceutical proteins in suspension cells. this makes their molecular farming products less expensive and more attractive. The ex vivo stability and in vivo efficacy of the plant and mammalian cell-culture produced antibodies were similar (Zeitlin et al. Zhong et al. 1998). which reached 7% of the total soluble protein. gastrointestinal. Higher expression levels will be reached by better control of gene silencing and the identification of novel.. ProdiGene (College Station. The decision on what spe- . It will also be valuable to screen a range of other plant species than used today for their use in molecular farming. Their interest is the production of human mucosal antibodies for passive immunisation by exploiting ProdiGene’s expertise in protein expression (Hood et al. Plant-produced antibodies are likely to allow development of an inexpensive method for mucosal immuno-protection against sexually transmitted diseases..5 tonnes (Table 5). 1999) together with EPIcyte’s academic and patent position. Middleton. A pharmaceutical partner plans to begin injecting cancer patients with doses of up to 250 mg of the antibodybased cancer drug purified from corn seeds. 1997.5 kg of pharmaceutical-quality protein per acre of corn. 1998. These antibodies were shown to be efficient in preventing vaginal HSV-2 transmission in mice. high level expression of many other pharmaceutical proteins may be possible and this approach is biologically selfcontained. Hood et al. respiratory. Wisconsin) created a corn line producing human antibodies at yields of 1. The researchers created a modified tobacco mosaic virus vector that encodes the idiotype-specific scFv of the immunoglobulin from the 38C13 mouse B cell lymphoma. 1998). CA) and Stanford University has developed a technology to produce a tumorspecific vaccine for the treatment of malignancies using a plant virus based transient expression system. These costs are often large. 1999. 1999). the bacteria that causes tooth decay in humans (Larrick et al. Vacaville. Infected Nicotiana benthamiana plants secreted high levels of scFv protein to the apoplast. is an exciting result (R. Kusnadi et al. The costs of licensing the technology for transforming plants or for using a promoter from its patent assignees are important aspects of molecular farming in plants. genital and urinary mucosal surfaces and skin. Monsanto (formerly Agracetus.html).. 2000). there is considerable room for improvement in yields. 1998).. Future directions The technical challenges that need to be solved for plants are essentially all related to expression levels of recombinant proteins. The collaborative research group at Biosource Technologies (now named the Large Scale Biology Corporation. Planet Biotechnology is also engaged in the design and development of novel sIgA-based therapeutics to treat infectious diseases and toxic conditions affecting oral. Witcher et al. Given that the yield per acre of corn is on the range of 3. Mice vaccinated with the affinity-purified 38C13 scFv generated > 10 µg/ml anti-idiotype immunoglobulins. These mice were protected from challenge by a lethal dose of the 38C13 tumor... Clearly. The recent report of high level expression of human growth hormone (somatotrophin) in tobacco chloroplasts. Planet Biotechnology has demonstrated that CaroRxTM can effectively eliminate Streptococcus mutans.prodigene. or better plant species as expression hosts.. The goal of the therapy is to create antibodies customized for each patient that will recognize unique markers on the surface of the malignant B-cells and target the cells for destruction. this plastid expression strategy may also be successful with mitochondria. close to the costs of development of the transgenic plant line or greater.com/news. personal communication. Bassuner. Staub et al. This antibody fragment reacted with an anti-idiotype antibody. suggesting that the plant-produced 38C13 scFv protein is properly folded. Texas) and EPIcyte Pharmaceuticals (San Diego) have entered into a strategic partnership to produce antibodies in corn (www.294 on sIgA antibodies produced in transgenic tobacco plants and is designed to prevent the oral bacterial infection that contributes to dental carries (Ma et al..

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