366
REVIEW ARTICLE
Journal of
Viral Vectors for Production of
Recombinant Proteins in Plants
CHIARA LICO,1 QIANG CHEN,2
AND
Cellular
Physiology
LUCA SANTI3*
1
UTS BIOTEC, Section of Genetics and Plant Genomics, ENEA CR Casaccia, Rome, Italy
2
Biodesign Institute and Department of Applied Biological Sciences, Arizona State University, Tempe, Arizona
3
Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy
Global demand for recombinant proteins has steadily accelerated for the last 20 years. These recombinant proteins have a wide range of
important applications, including vaccines and therapeutics for human and animal health, industrial enzymes, new materials and
components of novel nano-particles for various applications. The majority of recombinant proteins are produced by traditional biological
‘‘factories,’’ that is, predominantly mammalian and microbial cell cultures along with yeast and insect cells. However, these traditional
technologies cannot satisfy the increasing market demand due to prohibitive capital investment requirements. During the last two
decades, plants have been under intensive investigation to provide an alternative system for cost-effective, highly scalable, and safe
production of recombinant proteins. Although the genetic engineering of plant viral vectors for heterologous gene expression can be
dated back to the early 1980s, recent understanding of plant virology and technical progress in molecular biology have allowed for
significant improvements and fine tuning of these vectors. These breakthroughs enable the flourishing of a variety of new viral-based
expression systems and their wide application by academic and industry groups. In this review, we describe the principal plant viral-based
production strategies and the latest plant viral expression systems, with a particular focus on the variety of proteins produced and their
applications. We will summarize the recent progress in the downstream processing of plant materials for efficient extraction and
purification of recombinant proteins.
J. Cell. Physiol. 216: 366–377, 2008. ß 2008 Wiley-Liss, Inc.
Recombinant DNA technology was initially used to express
proteins that were difficult to produce in their native organisms.
Increasing efforts, however, have been focused on designing
new molecules with more desirable characteristics and/or
functionality. Pharmaceuticals and industrial enzymes were the
first recombinant biotech products on the world market and
biopharmaceuticals were the majority of commercialized
recombinant proteins (Pavlou and Reichert, 2004). Many
protein-based drugs, similar to traditional small molecule
pharmaceuticals, function as antagonists by binding to and
thereby inhibiting the activity of their target, such as an enzyme
or a receptor. Classical protein antagonists include full
monoclonal antibodies (mAbs), their single-chain derivatives
(ScFv) and mAb-fusion proteins. Recent research programs
have also focused on non-antibody antagonists that consist of
a scaffold protein displaying the inserted affinity peptide
(Walsh, 2006). Recombinant DNA technology also provided
an excellent alternative for developing safer vaccines. Subunit
vaccines are based on immunodominant protein components
of a pathogen, but do not contain its genetic material.
Consequently they cannot replicate, cause disease, or
introduce pathogens into non-endemic regions. Viral coat
proteins are exceptional subunit vaccine candidates and in
some cases are able to form virus-like particles (VLPs)
when expressed in heterologous systems. In fact, the only
recombinant subunit vaccines presently available are based on
VLPs. They are highly immunogenic and able to induce both
humoral and cellular responses (Chackerian, 2007).
In addition to the pharmaceutical industry, many other fields
are also relying intensely on recombinant proteins. Areas as
diverse as agro-food technology, chemistry, detergent
production, bioremediation, biosensoring, petroleum, and
paper industries all receive significant contribution from
applications of recombinant proteins. For example, increasing
needs for a diversity of food processing enzymes, for example,
amylase, lipase, xylanase, pullulanase and pectin modifying
enzymes, demand a substantial involvement of recombinant
protein technology (Olempska-Beer et al., 2006).
In the coming years, there will be a significant increase in
demand for high quality recombinant proteins. In response,
ß 2 0 0 8 W I L E Y - L I S S , I N C .
biological systems used for the production of proteins must be
scalable, cost-effective, safe and flexible enough to meet market
requirements. Current systems rely on ‘‘bio-factories,’’ that is,
mammalian, insect, yeast, and microbial cell cultures. The
majority of the recombinant proteins are currently produced in
Escherichia coli or mammalian cells with a few exceptions of
yeast or insect cells (Yin et al., 2007). All of these bio-factories
are based on fermentation technology of suspension cells in
bioreactors, which requires an enormous upfront capital
investment and, thereby, severely constrains their scalability.
The use of plants as production systems for recombinant
proteins has been actively investigated over the last two
decades. Plants are attractive as protein factories because
they can produce large volumes of products efficiently and
sustainably and, under certain conditions, can have significant
advantages in decreasing manufacturing costs (Hood et al.,
1999; Giddings, 2001). Plant systems are far less likely to harbor
microbes pathogenic to humans than mammalian cells or whole
transgenic animal systems. In addition, one of the major
advantages of plants is that they possess an endomembrane
system and secretory pathway that are similar to mammalian
cells (Vitale and Pedrazzini, 2005). Thus, proteins are generally
efficiently assembled with appropriate post-translational
modifications. These cost, scale, and safety advantages
make plant-made pharmaceuticals very promising for both
commercial pharmaceutical production and for manufacturing
products destined for the developing world.
Three approaches are commonly used to express
heterologous proteins in plants: (1) stable transformation of the
*Correspondence to: Luca Santi, Department of Biology,
University of Rome ‘Tor Vergata’, Via della Ricerca Scientifica
00133, Rome, Italy. E-mail: luca.santi@uniroma2.it
Received 12 December 2007; Accepted 22 January 2008
DOI: 10.1002/jcp.21423
PLANT VIRAL VECTORS
nuclear genome, (2) stable transformation of the chloroplastic
genome, and (3) viral transient transformation.
In stable transformation technology, an expression cassette
harboring the exogenous gene of interest is integrated into
the nuclear or plastid genome of plant cells, which results in the
acquired character to be stably inherited over generations.
These lines can be propagated vegetatively or by seed and thus,
readily scaled up for protein production. Stable nuclear
transformation is often achieved using Agrobacterium
tumefaciens, which delivers fragments of DNA into the plant
cell nucleus at random positions. Alternatively, the ‘‘biolistic’’
method (microprojectile bombardment) can be used for plant
hosts that are difficult to transform by Agrobacterium (Hansen
and Wright, 1999).
Due to their double membrane structure, chloroplast
transformation can be achieved only by bombardment with
tungsten or gold particles coated with fragments of DNA.
Stable transformation of the chloroplast offers several distinct
advantages in areas of transgene targeting, product yield, and
regulatory compliance. Since the chloroplast genome allows for
homologous recombination, transgenes can be precisely
targeted to a specific locus of the genome to avoiding positional
effect or accidental gene knock-outs. Each plant cell has several
chloroplasts and each of these contains many circular genomes.
Therefore, the shear copy number of the transgene enables
high-level expression of the target recombinant protein. Since
the chloroplast is inherited maternally, this technology reduces
the risk of potential transgene escape by pollen dissemination.
Similar to bacterial cells, however, the chloroplast is unable to
perform typical eukaryotic posttranslational modifications,
such as glycosylation. As a result, this technology cannot be
used to produce proteins when such modifications are essential
for their function (Bock, 2007).
The third strategy relies on replicating plant viruses. These
viruses are small, can be easily manipulated, and their infection
process is relatively simple. The above features make viral
vectors an attractive alternative to stable transgenic systems for
the expression of foreign proteins in plants. In this strategy, the
gene of interest is inserted among viral replicating elements,
episomically amplified, and subsequently translated in the plant
cell cytosol. Most of the transient expression systems are based
on non-food, non-feed plants like tobacco, therefore, requiring
further purification prior to application.
Production of recombinant proteins with stably or
transiently transformed plants has been performed both in
green-houses and in field. Neither practice requires large
investments in hardware or culture media, thus making
scale-up more economical than fermentation cultures. The
possibility of producing recombinant protein agents on an
agricultural scale by ‘‘molecular farming’’ is extremely
attractive.
Although the use of plants as expression systems for
recombinant proteins was first conceived for oral delivery of
antigenic proteins (Walmsley and Arntzen, 2000), plant-made
recombinant proteins have also been purified and used in many
different applications. An enormous number of proteins
different in size, structure, origin and biological function have
been successfully expressed in stably transformed monocot and
dicot plants such as maize (Chikwamba et al., 2003), rice (Nochi
et al., 2007), wheat (Brereton et al., 2007), potato (Youm et al.,
2007), tomato (Huang et al., 2005), tobacco (Watson et al.,
2004), lettuce (Sun et al., 2006), alfalfa (Huang et al., 2006), lupin
(Smart et al., 2003), carrots (Marquet-Blouin et al., 2003), barley
(Joensuu et al., 2006), soybean (Moravec et al., 2007), and thale
cress (Carrillo et al., 1998).
Many antigenic monomeric proteins from viruses (Webster
et al., 2006) and prokaryotes (Alvarez et al., 2006) have been
produced and in a few cases, their immunogenic properties
have been successfully evaluated in human volunteers during
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phase I clinical studies (Tacket et al., 1998, 2000; Thanavala
et al., 2005).
Complex oligomeric mAbs were first expressed in stable
transgenic plants by Hiatt et al. (1989). Since then, the number
and type of plant-expressed antibodies, such as secretory
antibodies, antibody fragments and, more recently, immune
complexes, have increased steadily. Several commercial
candidates of mAbs have been developed, with five mAbs under
clinical evaluation, and two having reached Phase II clinical
studies (Fischer et al., 2003).
In addition to pharmaceuticals, recombinant proteins for a
variety of applications have also been expressed in transgenic
plants. Examples include alpha-amylase enzyme (Pen et al.,
1992), the protein brazzein for commercial sweeteners
(Lamphear et al., 2005), and the fungal enzyme manganese
peroxidase (Clough et al., 2006). Moreover the chicken avidin
diagnostic protein (Hood et al., 1999) and the bovine trypsin
enzyme (Woodard et al., 2003) from transgenic maize, and the
human anti-microbic proteins lysozyme (Huang et al., 2002) and
lactoferrin (Humphrey et al., 2002) from transgenic rice have
been commercially produced and are currently marketed by
Sigma Chemical Company (St. Louis, MO).
The major challenges facing transgenic plant technology
lay in increasing the quantity of protein, the optimization
of expression systems, and improvement of downstream
processing. In this review, we will focus on transient production
strategies using plant viral expression systems, with a particular
focus on the variety of proteins produced, and their
applications. We will also discuss the latest developments in the
downstream processing of plant materials for efficient
extraction and purification of recombinant proteins.
Plant Viral Vectors
The history of genetic engineering and applied virology
are intimately connected; the first recombinant molecule
assembled was a chimeric SV40 containing genes from the
bacteriophage l (Jackson et al., 1972). The unique properties of
viruses such as ease of manipulation, high level amplification,
site specific recombination, strong infectivity, enhanced
translation and compact and repetitive morphological structure
have enabled their broad application, from basic research
to product development, including the generation of robust
expression systems. Viruses with a variety of host ranges such
as bacteriophages, mammalian retroviruses, invertebrate
infecting baculoviruses, and plant viruses have been genetically
modified to express heterologous proteins.
From the discovery of viruses in 1898 (tobacco mosaic virus,
TMV) (Bos, 1999), to the first demonstration of RNAs role in
virus replication by turnip yellow mosaic virus (TYMV)
(Matthews, 1989), to the very recent discovery of gene silencing
and its implication in host response to infection, gene regulation
and transgene expression (Baulcombe, 1999; Lu et al., 2003;
Waterhouse and Helliwell, 2003), plant virology has played a
crucial role in the understanding of the most fundamental
concepts of modern biology. In addition, plant viral elements
such as promoters, terminators, translational enhancers and
various cis-regulatory sequences have been extensively used in
plant biotechnology.
Plant viruses have been used to introduce foreign genes in
plants since the early 1980s and technical advances in molecular
biology and plant virology have allowed the generation of
many improved expression systems. These recent systems
provide many advantages including rapid, high-level transgene
expression, and, in the case of movement defective systems,
better transgene containment due to the lack of vertical and
horizontal gene transfer.
The earlier plant virus expression systems were based on the
cauliflower mosaic virus (CaMV) of the Caulimoviridae family,
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the only plant viruses with a double stranded DNA genome.
However, limited packaging capacity and the restricted amount
of viral DNA that can be removed without affecting essential
functions hindered the application of these initial expression
systems. Fortunately, technical advances in molecular biology
such as generating cDNA from an RNA template have enabled
us to expand our search for expression systems into single
stranded RNA viruses, the most represented types of plant
viruses in nature. Another technical breakthrough was the use
of Agrobacterium tumefaciens to promote viral infection
(Grimsley et al., 1986; Turpen et al., 1993). Generally, viral
infection can be initiated by mechanical inoculation of infectious
viral particles on leaves or alternatively by transfection of
nucleic acids. The use of Agrobacterium tumefaciens, known as
‘‘agroinfection,’’ allows the direct and efficient targeting of
cDNA to the plant cell nucleus (Fig. 1). The cDNA constructs in
the plant expression cassette would result in an infectious,
autonomously replicating nucleic acid after host cell nuclear
transcription and processing. Moreover agroinfection allowed
the use of viruses that in nature are not mechanically
transmissible, but instead need a special insect vector to initiate
the infection process. These viral vectors are commonly divided
into gene substitution vectors, gene insertion vectors, modular
or deconstructed vector systems and peptide display vectors.
Gene substitution or replacement vectors are based on the
exchange of an endogenous viral sequence with a heterologous
gene of interest. The first successful expression of a foreign
gene ever achieved in plants was obtained using a substitution
vector based on the CaMV. Usually, the coat protein (CP) gene
is the viral coding sequence of choice to be replaced. However,
with a few exceptions, the CP is essential for cell to cell and/or
systemic movement and infection. However, caution has to be
taken if leaves of the whole plants are intended to be infected.
For example, a CP replacement vector was developed using the
tomato bushy stunt virus (TBSV). In this case the CP is not
essential for systemic movement of the virus in certain species
of Nicotiana (Scholthof et al., 1993) and it was indeed
demonstrated that the insertion of a heterologous gene
supports a systemic infection (Scholthof et al., 1993). In
TMV-based substitution vectors, where the CP is necessary
for systemic infection, a chloramphenicol acetyl transferase
(CAT)–CP gene replacement vector generated only local
lesions and CAT activity was confined on the inoculated
leaf (Takamatsu et al., 1987). In another recently developed
TMV-based gene substitution vector (Musiychuk et al., 2007)
the CP gene was exchanged with the gene of interest fused to
a thermostable carrier molecule, the beta-1,3–1,4-glucanase
enzyme (lichenase) of Clostridium thermocellum, to facilitate
target expression, stability and purification. The heat tolerant
property of the lichenase (658C) allows easy target proteins
purification by heat treatment which precipitates up to 50% of
contaminating plant proteins. The protein of interest, fused at
the N- or C-terminus of lichenase, or inserted in the central
loop of an engineered version of the enzyme, is protected from
the degradation and thus purified.
Gene insertion vectors consist of complete functional
viruses with the addition of an extra open reading frame (ORF)
for the target protein. They are capable of cell to cell and
systemic movement and infection. Viruses with both spherical
and rod shaped virions have been investigated. Viruses with
rod-shaped particles have a better potential due to fewer
constraints on the amount of nucleic acid inserted. However,
there is still a limit on the genome size of the chimeric
rod-shaped virion based vectors. It has been proven that
chimeric vectors with genome size beyond certain limits
resulted in unsustained virion assembly. Two of the most
famous gene insertion vectors have been derived from TMV and
potato virus X (PVX). Both viruses have a single stranded
positive RNA genome. They use subgenomic promoters and
consequently subgenomic RNAs to express some of their ORFs
and have a helical virion symmetry which results in rod-shaped
particles. A chimeric TMV was constructed with the CAT gene
Fig. 1. Agroinfection method. Nicotiana benthamiana plant (A,B), leaves adaxial side (C) and abaxial side (D), Agrobacteriun tumefaciens liquid
culture (E), small scale infiltration procedure (F), GFP expression under U.V. light (G). [Color figure can be viewed in the online issue, which is
available at www.interscience.wiley.com.]
JOURNAL OF CELLULAR PHYSIOLOGY
PLANT VIRAL VECTORS
inserted between the movement protein (MP) gene and the CP
gene; the expression was regulated by an additional copy of the
subgenomic promoter of the CP gene that, as a result, was
duplicated in the hybrid vector. The vector was able to
replicate, to form subgenomic RNAs, to assemble correctly,
and to produce reporter gene activity. Nevertheless the
duplication of the subgenomic promoter sustained homologous
recombination causing instability and the consequent loss of the
exogenous gene (Dawson et al., 1989). This problem was solved
by using a subgenomic CP promoter derived from a different
virus belonging to the Tobamovirus genus to prevent
homologous recombination. Similar problems and solutions of
recombination between duplicated subgenomic promoters
were reported in a recent insertion vector system based on the
grapevine virus A (GVA) for N. benthamiana (Haviv et al., 2006).
Analogous to TMV, a PVX-based vector was generated using
the duplicated promoter of the CP gene. However in this case
the vector was stable and able to retain the additional coding
sequence (Chapman et al., 1992). Nevertheless, recently it has
been demonstrated that the size of the inserted gene of interest
can play a crucial role in the stability of the modified PVX
expression vector, with a positive correlation between the
elimination rate of the gene of interest and its length (Avesani
et al., 2007). Recombination can involve homologous CP
promoter sequences but also other mechanisms mediated by
both the host plant and the virus (Avesani et al., 2007).
Another well established system is based on Cowpea Mosaic
Virus (CPMV) the type member of Comoviridae whose bipartite
genome is constituted by single stranded positive RNA
molecules. RNA-1 encodes for proteins involved in replication
while RNA-2 carries the genetic information for the movement
and structural proteins. The system is very versatile; it can be
utilized in a totally transient fashion, co-infiltrating wild-type
plants with two separate c-DNA constructs representing
RNA-1 to sustain amplification and RNA-2 harboring the gene
of interest, or it can be utilized combining transgenic plants with
virus-mediated transient expression. In this case the RNA-2
is used for stable transformation and the RNA-1, necessary to
prime the active replication of RNA-2 and of the heterologous
gene, is supplemented by agro-infiltration. It is worth noticing
that a deleted version of RNA-2, unable to produce infectious
clones, it has also been utilized to prevent possible
environmental containment concerns (Liu et al., 2005;
Sainsbury et al., 2008).
In conclusion it is possible to obtain an inducible expression
system if RNA-1 is supplemented by agroinfiltration and a
constitutive stable expression system if it is supplemented by
crossing with an RNA-1 stable transgenic line (Canizares et al.,
2006).
Modular or deconstructed vectors are a new generation of
viral expression systems. Their development was driven by the
understanding that: (1) not all viral components are essential
or beneficial for an expression vector and (2) viruses can be
broken down into different genomic elements that would
still operate together in the infection process as wild-type
multipartite genome viruses. Moreover, agroinfection provided
the technical possibility to co-deliver multiple different
components. Hence in modular systems, viral components
are separated into distinct portions and inserted into binary
vectors contained in an Agrobacterium strain. The strains are
mixed together and co-infiltrated into plant leaves. This
strategy allows the replicative portion, the so called replicon,
to be reduced in size, down to minimal to accommodate the
insertion of transgene, while other viral components necessary
for the vector function can be provided in trans during the
infection process. At present, one of the most famous and
broadly used vector is the deconstructed system based on TMV
and developed by Icon Genetics (Halle, Germany), recently
acquired by Bayer Innovation GmbH. The vector has been
JOURNAL OF CELLULAR PHYSIOLOGY
engineered to divide the TMV genome into two major cDNA
modules: a 50 module which contains the viral RNA dependent
RNA polymerase and the MP, and a 30 module that carries the
gene of interest and the 30 untranslated region (UTR) of the
virus essential for the efficient replication and amplification
of the vector. In this particular case, viral functions are not
complemented in trans but the two modules actually assemble
together in vivo by a site specific recombinase delivered by a
third Agrobacterium cell line. Furthermore, different 50 modules
carry different organelle targeting signals which, fuse in frame
with the gene of interest after recombination and nuclear
processing, allow a single construct to be combined pair wise
with various targeting elements in separate constructs. This
feature greatly facilitates the experimental procedure to test
optimal expression in terms of accumulation in different
sub-cellular compartments. An additional attribute of the
vector, that permitted a dramatic enhancement of expression
levels, was the introduction of several introns in the coding
sequences of the 50 module (Marillonnet et al., 2004, 2005).
Levels of expression are impressive and can reach up to 5 mg
of recombinant protein per gram of fresh weight (FW). The
system was extensively tested with different genes, coding
for antibodies and antibody-derivatives, interferons, growth
hormones, bacterial and viral antigens, adjuvants and enzymes
(Gleba et al., 2005), confirming each time its versatility and
robustness.
Another interesting modular system was developed using
the bean yellow dwarf virus (BeYDV) of the Geminiviridae family.
Geminivirus are single stranded circular DNA viruses known
to replicate through the rolling circle process, promoting high
level of genome amplification in the nucleus of the infected cell.
The BeYDV based system is depleted of the MP and CP gene
and consequently the expression of the protein of interest is
confined to the inoculation area. The basic version involves two
modules, one containing the gene of interest inserted between
the BeYDV long and short intragenic regions, LIR and SIR
respectively. Both intragenic regions are necessary to sustain
rolling circle replication. The gene of interest is driven by a
strong plant specific constitutive promoter. The second
module is responsible for the expression of the Rep protein
which catalyzes various aspects of the rolling circle replication.
Initially, the system was used as an expression vector in tobacco
plant cell culture (Mor et al., 2003) but its use has recently been
extended to whole leaves with agroinfection. The modular
nature of this system also permits the co-expression of a variety
of sequences of different viral or non-viral origin that are
beneficial for replicon amplification and transgene expression.
For example, genes of post-transcriptional gene silencing
(PTGS) inhibiting proteins can be co-expressed to prevent
PTGS and hence enhance target protein accumulation.
Display viral vectors have been extensively used to provide a
molecular scaffolding for different kinds of peptides to be fused
with the viral CP and, therefore, to be exposed and displayed on
the surface of chimeric virus particles (CVPs). Coat protein
genes of several plant viruses were genetically modified to
support fusions with coding sequences of heterologous
peptides in specific regions known to be well exposed on the
virion surface (Johnson et al., 1997; Porta and Lomonossoff,
1998). Therefore, the virus and the plant host would provide
the expression system to produce large quantities of the
chimeric fusion protein. Clearly this is only feasible for well
characterized viruses in terms of assembly, movement
strategies and structure. Fusions must be compatible with the
normal assembly and viral fitness, and must avoid any possible
steric hindrance or interference with virus movement. Defining
peptide features for their correct display on CVP is of particular
interest among studies in this area (Bendahmane et al., 1999;
Porta et al., 2003; Lico et al., 2006). Two strategies have been
developed to overcome assembly problems for long or difficult
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peptides. One strategy is to modify the 30 terminus of the CP
gene so that the CP stop codon would function in a leaky
fashion. This would cause most of the CP produced to be
unfused, while a small portion would be fused to the peptide
displayed on the surface (Hamamoto et al., 1993; Sugiyama
et al., 1995; Borovsky et al., 2006). The other strategy employs
the 2A peptide of the small foot and mouth disease virus
(FMDV). This sequence, inserted between the epitope coding
sequence and the 50 terminus of the CP gene, produces a
ribosomal skip during the translational procedure (Donnelly
et al., 2001; de Felipe et al., 2003), with the result that only a
small portion of the translational products will consist in a
fusion between the heterologous sequence and the CP (Fig. 2).
The use of these strategies, however, opens the question of the
precise dose of the antigen in the vaccine formulation. Many
plant viruses have been used to display different kinds of
peptides. The most widely used viruses for this purpose are the
CPMV, the TMV and the PVX. Extensive research regarding the
symmetry, shape and structure at atomic level for these viruses
have allowed the precise definition of specific sites for peptide
insertion to ensure the successful display of fusion peptides
(Klug and Caspar, 1960; Lomonossoff and Johnson, 1991;
Baratova et al., 1992a,b; Johnson et al., 1997; Parker et al., 2002).
Major efforts are now directed to the development of new
viral vector typologies in terms of both improvement of the
expression strategies and virus choice. Host plants have also
been extended to a wider variety of species including
herbaceous plants and cucurbitaceous family. Besides the
viruses mentioned above, other viruses have also been
investigated as useful vectors, such as tomato mosaic virus
(ToMV) (Dohi et al., 2006); maize streak virus (MSV) (Palmer
and Rybicki, 2001); bean pod mottle virus (BPMV) (Zhang and
Ghabrial, 2006); beet necrotic yellow vein virus (BNYVV)
(Schmidlin et al., 2005); tomato golden mosaic virus (TGMV)
(Hayes et al., 1989); potato virus A (PVA) (Kelloniemi et al.,
2006); zucchini yellow mosaic virus (ZYMV) (Hsu et al., 2004);
cucumber mosaic virus (CMV) (Nuzzaci et al., 2007); and
cucumber green mottle mosaic virus (CGMMV) (Ooi et al.,
2006).
Monoclonal antibodies
Antibodies are key molecules of the vertebrates’ immune
system. They are responsible for the recognition and binding of
target antigens with high affinity and specificity. Monoclonal
antibodies (mAbs) are used in a wide range of applications,
Fig. 2. Display strategies. Wild-type viral genome organization and encoded proteins (a), standard CP fusion strategy (b), Foot and Mouth
Disease Virus 2A peptide CP fusion strategy (c), leaky stop codon CP fusion strategy (d). RdRp, RNA dependent RNA polymerase; MP, movement
protein; CP, coat protein; 2A, Foot and Mouth Disease Virus 2A peptide; LSC, amber leaky stop codon. [Color figure can be viewed in the online
issue, which is available at www.interscience.wiley.com.]
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PLANT VIRAL VECTORS
including diagnosis, prevention and treatment of many diseases.
For example, they are being successfully utilized as ‘‘targeting
vectors’’ to direct drugs, radioisotopes and other therapeutic
molecules to their target cells or tissues, as immunomodulators
in pathology-related functions, as well as diagnostic tools to
identify and localize molecular alterations. In addition to their
pharmaceutical applications, mAbs can also be used to
modulate plant traits, generate disease or pest resistant crops
(Tavladoraki et al., 1993; Nolke et al., 2004; Peschen et al., 2004;
Villani et al., 2005), and to study plant metabolic pathways
(Nolke et al., 2005). Antibodies are complex glycoproteins
consisting of four polypeptides, linked by disulfide bridges and
non-covalent bonds. Despite their complexity, mAbs and their
derivatives can be easily expressed in heterologous systems
including plants (Fischer et al., 2003). However, mAbs
produced by heterologous systems may not share the precise
structural and functional properties of the native molecules. For
example, mAbs produced by a bacterial expression system will
lack post-translational modifications including glycosylation.
Non-mammalian eukaryotic expression systems could fail to
use the appropriate glycans groups resulting in different
glycosylation patterns. Yeasts, for example, tend to
hyperglycosylate (Harashima, 1994; Malissard et al., 1996);
while plants preferentially introduce xylose and fucose residues
and fail to use galactose and syalic acid (Gomord and Faye,
2004). Although proper glycosylation is required for functions
of many mAbs, it is to be noted that different glycosylation
patterns do not always lead to loss of function in vitro or in vivo,
or cause side-effects in humans (Chargelegue et al., 2000).
Different strategies have been adopted to circumvent these
limitations such as targeting mAbs to the endoplasmic
reticulum, or abolishing glycosylation sites by mutagenesis to
avoid inappropriate glycosylated products. In fact, it has been
reported that the absence of glycans in some cases did not
interfere with correct assembly and activity (Rodriguez et al.,
2005; Nuttall et al., 2005). In addition a fascinating approach that
requires sophisticated genetic and metabolic engineering
resides in the inactivation of endogenous glycosyltransferases
and/or expression of heterologous mammalian specific
enzymes in the host plant system (Saint-Jore-Dupas et al.,
2007).
Complete antibodies and their derivatives have been
produced with stable plant transformation technologies (Ma
et al., 2005). Thanks to the recent improvements of viral-based
vectors, mAbs have been produced with transient expression
systems to quickly achieve much higher production levels along
with other complex proteins. Single chain fragments, that
preserve antigen-recognition elements of the full length
immunoglobulin, are often more effective as drugs or diagnostic
tools than mAbs due to their increased ability to penetrate
target tissues, reduced immunogenicity, and more rapid
clearance. A chimeric gene insertion TMV based vector was
used for the expression of a human scFv for treatment of
non-Hodgkin’s lymphoma (NHL) (McCormick et al., 1999,
2003). The plant-derived scFv generated an antibody response
in vaccinated animals, and was an effective vaccine in a murine
NHL tumor challenge model. The TMV strategy provides
the speed and versatility required for the development of a
patient specific treatment and this anti-idiotype vaccine is
currently undergoing clinical trials for safety and
immunogenicity evaluation in humans.
Another example is a scFv derivative (small immune protein,
SIP) to treat transmissible gastroenteritis virus (TGEV), a
porcine coronavirus. The SIP was expressed by a PVX gene
insertion vector and by a CPMV based display vector using
the 2A expression strategy. The aim was to provide passive
protection against enteric infections upon oral delivery in crude
plant extracts (Monger et al., 2006). The SIP folded correctly
and preserved the ability to bind and neutralize TGEV in tissue
JOURNAL OF CELLULAR PHYSIOLOGY
cultures. Moreover, it provided in vivo protection against
challenge with TGEV in piglets vaccinated orally with crude
extracts of scFv-expressing tobacco plants. The same
researchers also expressed a full length TGEV-specific IgA by
co-infecting plants with two separate PVX insertion vectors for
the light chain (LC) and the heavy chain (HC) respectively
(Alamillo et al., 2006). The IgA was correctly assembled in plant
tissue and provided in vivo protection against TGEV in piglets
after oral administration. A previous work (Verch et al., 1998)
also indicated the possibility to produce a full size mAb in plants
through the co-infection of two modified TMV based viral
vectors. These results clearly indicated the possibility to obtain
functional mAbs through plant expression systems.
Nonetheless, expression of heterooligomeric proteins such
as mAbs might be inefficient due to the co-delivery of viral
vectors built on the same virus backbone. In fact, this feature
may result in early segregation and subsequent preferential
amplification of one of the vectors in one cell. This problem has
been recently overcome by utilizing two sets of vectors derived
from non-competing TMV and PVX to express the two
different mAb chains (Giritch et al., 2006). TMV and PVX may
interact with different host proteins to sustain their replication
and movement without interfering with each other. As a result,
neither of the two vectors gains a replicative advantage over
the other, allowing efficient co-expression of HC and LC in
the same cells. The level of expression by each vector is
independent within the same cell and can reach up to 0.5 mg/g
FW of full assembled mAb in few weeks. This strategy
represents a useful platform for rapid large-scale manufacturing
of mAbs and other hetero-oligomeric proteins especially in
situations requiring rapid responses such as pandemic threats
and bioterrorism events.
Antigens
A vaccine is an antigenic preparation used to establish immunity
to a disease. Vaccination has been one of the greatest
revolutions in medical science and has dramatically improved
the quality and length of life expectancy. Although vaccines are
the most cost effective form of disease protection in healthcare,
their cost is still too excessive for many people in the world,
especially in developing countries. Vaccines can be therapeutic,
to overcome an infection already established, or prophylactic,
to prevent a future infection. The aim of vaccination strategies is
to obtain efficient and safe vaccine formulations to induce a long
lasting immunity against various pathogens. To achieve this goal,
the vaccine component should generate not only a neutralizing
antibody response, a long-term memory B cells stimulation, but
also a T cell mediated immunity to eliminate infected cells which
represent the pathogen reservoirs. Recombinant subunit
vaccines, based on a single protein (antigen) or a single peptide
(epitope) derived from the pathogen, are particularly attractive
strategies when compared to classic inactivated, attenuated or
live recombinant vaccines. Recombinant subunit vaccines offer
potentially equivalent efficacy but are much safer and in some
cases easier to produce. Plants offer unique advantages for the
production of subunit vaccines in terms of scale, speed, costs,
yield, and safety. The first work that describes the expression of
the hepatitis B surface antigen in stable transgenic tobacco in
1992 marked the beginning for developing low cost vaccine
formulations in plants (Mason et al., 1992). Researchers focused
on expressing their antigens in edible plant tissues as oral
vaccines, opening a new prospect for vaccine delivery (Mor
et al., 1998). Since then, many research groups have adopted
this system and carried out studies in the areas of improving
target protein expression levels, analyzing the administration
route and schedule, choosing the appropriate animal models,
and exploring the use of possible adjuvants.
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The first report in this area described the expression of the
FMDV structural protein VP1 with a TMV-based gene insertion
vector (Wigdorovitz et al., 1999). A yield of approximately
0.15 mg/g FW was achieved and subsequently the antigen was
administered to mice without further purification. All mice
immunized intraperitoneally developed a protective immune
response against experimental challenge with virulent FMDV. A
great number of antigens have been produced in plants with
different immunologic results (Streatfield and Howard, 2003).
Altogether, these works demonstrated that plant-derived
antigens retained their antigenicity and were able to induce
active protective humoral and cell-mediated immune
responses. The development of plant derived vaccines for
human and veterinary uses against viral pathologies, tumors,
allergies, bacterial infections, and diabetes-associated
autoantigens has been extensively investigated (Table 1).
Yusibov et al., expressed the E7 oncoprotein from human
papilloma virus (HPV) in N. benthamiana plants with a gene
replacement vector. The antigen was subsequently purified
from infected leaves using the thermotolerance properties of
lichenase (Massa et al., 2007). Mice, immunized subcutaneously
with the plant-produced E7, showed a specific antibody and
cytotoxic T-cell response. The dose also protected the animals
against challenge with E7 tumor cells. Moreover, the
recombinant antigen was able to prevent tumor development
when administered after virus challenge, supporting the
ultimate goal to produce an anti-tumor vaccine with both
therapeutic and prophylactic uses.
A fundamental revolution within the last few years came
from the use of modular deconstructed systems. A recent work
using the magnICON TMV based system is worth noting. Santi
et al. (2006) focused on the production of antigens for agents of
biological warfare, for example, the Yersinia pestis bacterium,
the causative agent of plague. In this work the protective
efficacy of the fraction 1 capsular antigen (F1) and the low
calcium response virulent antigen (V) expressed at levels up to
2 mg/g FW in N. benthamiana leaves was evaluated. Guinea pigs,
immunized with purified antigens, showed antigen-specific
serum IgG titers and, more importantly, 75% of animals were
protected from an aerosolized challenge of virulent Y. pestis. In a
subsequent work Mett et al. (2007) expressed the same Y. pestis
F1 and V antigens in N. benthamiana plants with an average
yield of 0.38 mg/g FW with their lichenase optimized vector.
The purified proteins were administered subcutaneously to
non-human primates, Cynomolgus Macaques, producing
antigen specific IgG and IgA responses which resulted in
complete protection against lethal challenge with Y. pestis.
Recent work show a particular interest on emerging and
re-emerging diseases, as well as agents of biological warfare, and
have extended plant-derived vaccine development to smallpox
(Golovkin et al., 2007), anthrax (Chichester et al., 2007),
dengue virus (Saejung et al., 2007), and avian influenza A virus
(Nemchinov and Natilla, 2007). Altogether, these data
collectively show the establishment of a portfolio of well
characterized and novel, improved strategies for the
expression of effective vaccines in plants.
Viral display for vaccine formulations
‘‘Peptide’’ vaccines are a particular subclass of subunit vaccines
based on the ability of immunodominant epitopes to induce
specific immune responses. Epitopes are the most important
regions of an antigen. They interact directly with surface
receptors on B and T cells and induce an immune response.
A single protein can have several epitopes with different
immunological features and structures. Epitopes can be linear,
TABLE 1. Representative antigens expressed through viral vectors
Pathogen
Antigen
Viral vector
References
Human immunodeficiency virus-1
Plasmodium falciparum
Influenza virus
Human immunodeficiency virus-1
Capsid epitopes
Several epitopes
HA epitope
Capsid epitopes
Mink enteritis virus
Canine parvovirus
Staphylococcus aureus
Foot and mouth disease virus
Hepatitis C virus
Rotavirus
Human immunodeficiency virus-1
Pseudomonas aeruginosa
Human immunodeficiency virus-1
Rabbit haemorrhagic disease virus
Rabies virus
Human papilloma virus
Bovine herpes virus
Hepatitis C virus
Colorectal antigen
Human immunodeficiency virus-1
Respiratory syncytial virus
Classical swine fever
Canine oral papillomavirus
Melanoma
Yersinia pestis
Influenza A virus
Dengue virus
Mycobacterium tuberculosis
Human papilloma virus
Smallpox
Yersinia pestis
Bacillus anthracis
Bacillus anthracis
VP2 protein peptide
VP2 protein peptide
FnBP epitope
VP1 protein
Region 1 of E2
VP6 protein
P24 protein
F protein peptides
Capsid epitopes
VP60 protein
Chimeric peptide
E7 oncoprotein
Glycoprotein D
Mimotope
GA733-2 antigen
Tat protein
G protein epitope
E2 glycoprotein peptides
L2 protein
p15e-Trp2 epitopes
F1 and V proteins
M2 protein ectodomain
Domain III of E protein
ESAT6—Ag85B antigens
E7 protein
pB5 antigenic domain
F1 and V proteins
PA peptide
PA and LF domains
CPMV (D)
TMV (D)
TMV (D)
AMV (D)
TBSV (D)
CPMV (D)
PPV (D)
CPMV (D)
TMV (I)
TMV (I)
PVX (D)
TBSV (I)
TMV (D)
PVX (D)
PPV (I)
AMV (D)
PVX (I)
TMV (I)
CMV (D)
TMV (I)
TMV (I)
AMV (D)
PVX (D)
TMV (D)
TMV (D)
TMV (M)
PVX (I)
TMV (I)
TMV (I, D)
TMV (S)
TMV (M)
TMV (S)
CPMV (D)
TMV (S)
Porta et al. (1994)
Turpen et al. (1995)
Sugiyama et al. (1995)
Yusibov et al. (1997)
Joelson et al. (1997)
Dalsgaard et al. (1997)
Fernandez-Fernandez et al. (1998)
Brennan et al. (1999)
Wigdorovitz et al. (1999)
Nemchinov et al. (2000)
O’Brien et al. (2000)
Zhang et al. (2000)
Staczek et al. (2000)
Marusic et al. (2001)
Fernandez-Fernandez et al. (2001)
Yusibov et al. (2002)
Franconi et al. (2002)
Perez Filgueira et al. (2003)
Natilla et al. (2004)
Verch et al. (2004)
Karasev et al. (2005)
Yusibov et al. (2005)
Marconi et al. (2006)
Smith et al. (2006)
McCormick et al. (2006b)
Santi et al. (2006)
Nemchinov and Natilla (2007)
Saejung et al. (2007)
Dorokhov et al. (2007)
Massa et al. (2007)
Golovkin et al. (2007)
Mett et al. (2007)
Phelps et al. (2007)
Chichester et al. (2007)
CPMV, cowpea mosaic virus; TMV, tobacco mosaic virus; AMV, alfalfa mosaic virus; TBSV, tomato bushy stunt virus; PPV, plum pox virus; PVX, potato virus X; CMV, cucumber mosaic virus; I,
insertion vector; S, substitution vector; M, modular/deconstructed vector; D, viral display.
JOURNAL OF CELLULAR PHYSIOLOGY
PLANT VIRAL VECTORS
determined by the amino acid sequence, or conformational,
determined by neighboring amino acids only in the
tridimensional tertiary structure. An epitope alone could be
poorly immunogenic and often possess a short half-life in the
serum (Lien and Lowman, 2003). As a result, extensive efforts
are being directed to the development of new delivery
strategies to increase the immunogenicity and half-life of
epitope vaccines (Purcell et al., 2007). Plant viral display vectors
have the potential to play an important role in such strategic
development. Plant virus particles can structurally function as a
scaffold to support, stabilize and display epitopic peptides. For
example, a target epitope can be genetically fused to the CP
protein. The chimeric virion will be formed by the CP-epitope
fusion, representing the vaccine by itself. As a result, plant
viruses can be used as a carrier to stabilize epitopes and to
present them correctly to the immune system.
Strategies such as the use of the amber leaky stop codon and
2A peptide (described earlier) have been adopted successfully
to display peptides (Turpen et al., 1995; Marconi et al., 2006), as
well as for full-length protein fusions (O’Brien et al., 2000).
In addition, new strategies involving biotinylation of the capsid
and the subsequent binding of streptavidin-conjugated target
protein have been developed for displaying long sequences. The
canine oral papillomavirus L2 protein, displayed on the TMV
surface with this strategy, was significantly more immunogenic
in animal models compared to the uncoupled antigen (Smith
et al., 2006). With this strategy, it is also possible to combine
helper epitopes and peptides known to facilitate cellular uptake
or other additional T-cell targets to avoid the use of adjuvants.
Coexpression of melanoma-associated cytotoxic T
lymphocytes (CTL) epitopes p15e and Trp2 on a TMV scaffold
stimulated effective tumor protection from challenge and
showed a significant survival improvement over the single
peptides alone (McCormick et al., 2006a).
The capability of CVPs in inducing an antibody response
specifically to the displayed epitope upon intranasal,
intraperitoneal or oral administration, have been extensively
demonstrated in different animal models (Table 1). Other data
show CVPs ability to elicit a strong specific neutralizing immune
response in the absence of adjuvants (Yusibov et al., 1997;
Brennan et al., 1999; Marusic et al., 2001). Oral delivery of
spinach leaves infected with CVPs elicited a strong antibody
response to the displayed rabies virus peptide in mice and
human volunteers (Yusibov et al., 2002), indicating a potential
use of this system as a supplementary oral booster for rabies
vaccinations. Alfalfa mosaic virus (AMV)-derived CVPs were
able to elicit T- and B-cell responses, in non-human primates
(Yusibov et al., 2005). Chimeric TMV particles was shown to be
able to directly interact with and stimulate mammalian antigen
presenting cells to induce a strong cellular anti-tumor immune
response (McCormick et al., 2006b). Studies of body
distribution for orally administered CVPs also suggested their
application as nanocapsules in novel drug delivery systems
(Rae et al., 2005; Smith et al., 2007).
Applications of peptide-displaying CVPs extend far beyond
the area of vaccine development. Of particular interest are a
killer peptide to confer broad-spectrum resistance to
phytopathogens (Donini et al., 2005), a metal-binding peptide
converting CVPs into an artificial metal-adsorbing sink for metal
tolerance and phytoremediation (Shingu et al., 2006), and a
mosquito hormone-derived decapeptide as a larvicide to
protect plants against agricultural insect pests and to control
vector mosquitoes (Borovsky et al., 2006). Finally Werner
et al. (2006) fused a fragment of protein A (133aa) to the TMV
CP C-terminus via a 15-aa linker. This chimeric nanoparticle
allowed a simple purification of mAbs with 50% recovery yield
and product purity of greater than 90%. This technology
provides an inexpensive self-assembling matrix that could be
used as industrial immuno-adsorbent for antibody purification.
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Nanotechnology applications
Due to their simple macromolecular organization, assembly
capabilities, structure stability, easy scalability and facile
purification, plant viruses can offer a cheap source of
biopolymers and nanoparticles. Well characterized viruses with
stable, highly ordered and repetitive structures, such as TMV
and CPMV, are of particular interests. For example, CPMV,
cowpea chlorotic mottle virus (CCMV) and other icosahedral
viruses have been used as nucleation cages for the
mineralization of materials (Douglas and Young, 1998).
Another promising application is in noninvasive in vivo vascular
imaging techniques (Manchester and Singh, 2006). CPMV for
example has been fluorescently labeled to visualize blood flow
for periods of at least 72 h to identify vessels and to monitor
tumor neovascularization (Lewis et al., 2006). Moreover,
CPMV can be modified to generate new surface properties for
developing novel biomaterials, electrochemical biosensors, or
nanoelectronic devices (Wang et al., 2002; Chatterji et al., 2004;
Steinmetz et al., 2006). Rod-shaped viruses, like TMV, have
been extensively used in the synthesis of a variety of metals
nanowires, magnetic materials and semiconductors. TMV can
be used as organic templates for the controlled deposition of
gold, silver and platinum to prepare 1-D arrays (Dujardin et al.,
2003). Self-assembled, modified CP monomers of TMV were
also employed for the construction of photovoltaic
components in a novel light-harvesting system (Miller et al.,
2007). CVPs can be also incorporated in liquid crystal systems
or used to generate thin films and fibers (Flynn et al., 2003).
These examples clearly demonstrate the broad application and
emerging potential of plant viruses in nanotechnology fields.
Downstream Processing of Plant-Derived
Pharmaceuticals
Over the last decade, plant based production of
pharmaceuticals has made remarkable progress as the
expression of a diverse set of proteins has been demonstrated,
several proteins have moved into clinical testing, and a
plant-derived veterinary vaccine has been approved. Recent
developments in transient expression systems and production
in a controlled green house environment are directly
addressing the issues of low expression levels and under
developed regulatory standards for plant made pharmaceuticals
(PMPs).
In spite of this progress, barriers remain that prevent the
broad adoption of this technology platform. One of these is the
lack of translational research to bridge the gap between bench
discoveries and their corresponding clinical products. In fact,
many product failures during development are ultimately
caused by problems of transition from laboratory prototype to
industrial product, as stated by the FDA Critical Path Report
(http://www.fda.gov/oc/initiatives/criticalpath/). Product
industrialization programs are routinely delayed or derailed
by inadequate efforts in downstream manufacturing, scale-up
development, and quality control. Therefore, as tremendous
progress has been realized in recombinant protein expression,
research focus has gradually been shifted to improvements in
downstream processes including extraction, purification and
recovery of the final products. Downstream processing is
fundamentally important to the commercial viability of the
specific PMPs and the PMP technology in general. An optimized
downstream process will not only provide the additional
cost-saving measures for the overall product cost, it will also
enable the large-scale production of the products to meet
the market demands. In addition, downstream process
development is an essential step in compliance with FDA’s
current Good Manufacture Practice (cGMP) regulations.
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The general downstream processing steps for extraction and
purification of pharmaceutical proteins are similar to those of
other systems. These steps include harvest and fractionation of
the tissue containing the targeted protein, extraction of target
protein into designed buffer, clarification of cell debris and
particles from the extract, purification of the target protein, and
vialing of the purified products into the proper container in the
desired formulation buffer. The unique structure and
biochemistry of plant cells and tissues present different
challenges and opportunities for each of the above processing
steps. In addition, the choice of host plant species, tissue and
subcellular organelle targeting of the product will have
profound impact on the strategy and outcome of downstream
processing efforts. Since this review concerns primarily viral
vector based transient production of PMPs, our discussion will
focus on the downstream bioprocessing of proteins targeted to
the cytoplasm of leafy plants. PMP production with seeds has
been extensively reviewed by others (Menkhaus et al., 2004;
Nikolov and Woodard, 2004).
Biomaterial harvest and fractionation
The first step of biomaterial processing is extraction and
purification from tissue with a high concentration of the target
recombinant protein. The selective harvest of the target tissue
will reduce the volume of initial biomaterial, and in turn, reduce
the unnecessary need to eliminate the extra host proteins
introduced by the inclusion of non-target tissue, and the total
operational cost. The selectivity will also allow the avoidance of
tissues which are hard to process, prime to introducing
environmental contaminants (e.g., roots), or rich in other
undesirable molecules such as proteases, alkaloids, and
phenolics.
For transient expression with viral vectors, leaves are
primarily the target tissue for protein accumulation. Currently
most of the biomaterial production with these vectors is
accomplished in greens houses with a controlled environment.
For example, 4–18 days after inoculation, N. benthamiana leaves
will be selectively harvested away from roots and stems
(Werner et al., 2006). For small scale preparation, some
researchers perform further fractionation of leaves by manually
separating the central vein from the rest of leaves. For largescale production, this kind of operation becomes impractical
and whole leaves are processed thoroughly in the next step. If
the biomaterial is produced in open fields, extra steps have to
be performed to remove the diverse array of environmental
contaminants. Rinsing and light washing of plants, before
harvesting the target tissue, is a common practice for this
purpose. Regardless of where the biomaterial is generated, it is
important to lower the bioburden and other contaminants as
much as possible at this step. This measure will prevent
microorganisms and other contaminants from entering
purification feed streams and thus, greatly simplify the
subsequent purification process and ensure the regulatory
compliance of the final product.
One of critical factors in choosing plant expression platforms
is the protein stability in the targeted tissue. With all the
advantages of the viral based transient expression system,
recombinant proteins generally do have less long-term stability
in leaves due to higher water content in contrast to the seedbased stable transformed expression systems (Doran, 2006).
However, some proteins did show relative long-term stability in
leaves of up to 7 days at room temperature (Fiedler et al., 1997)
or even longer (12 weeks) in dry alfalfa tissue (Khoudi et al.,
1999). Depending on the nature of the target protein, some of
them need to be further processed as fresh leaf tissue, while
others can be stored up to months as frozen tissue without
losing their recovery and/or biological activity (Chen,
unpublished work). The possibility of storing tissue at a cold
JOURNAL OF CELLULAR PHYSIOLOGY
temperature for certain proteins offers and expands the
flexibility of this production platform.
Protein extraction
The major goal of this processing step is to release the target
protein into a liquid buffer solution from the plant tissue. To
achieve this goal in a leaf-based transient expression system, leaf
tissue is first ground in the presence of a desired extraction
buffer to break the tissue and cells. The tissue homogenate will
then be further pressed to release the protein into the aqueous
buffer. The crude extract will be clarified to separate the
protein-containing buffer from plant debris, insoluble proteins
(see below) and other particulate matters. In comparison to
other production hosts, plants produce more solid debris (up
to 30% of totally weight of the biomaterial (Menkhaus et al.,
2004)). The larger and denser solids make it impossible to
achieve effective clarification by filtration methods. As a result,
continuous centrifugation remains the most effective and
scalable technique for plant extract clarification. It should be
noted that it is possible to extract small recombinant proteins
(<50 kDa) that are targeted through the endomembrane
system to the apoplast without breaking the plant cells. For
example, some of the smaller proteins can be released directly
into the extraction buffer by simply rinsing the tissue or by a
specialized centrifugation technique (Lohaus et al., 2001). The
purification process for this class of proteins can be greatly
simplified with the result of a reduction in host protein
contaminants.
The choice of extraction buffer has to be carefully
determined based on the properties (e.g., pI, size,
hydrophobicity, and stability) of the target protein and the
major contaminating host molecules. For example, the major
contaminating protein in plant leaves is ribulose 1,5bisphosphate carboxylase-oxygenase (RuBisCo).
Correspondingly, the choice of low pH buffers (pH 5.3)
should be encouraged to keep RuBisCo insoluble and prevent it
being extracted into the aqueous phase. At laboratory bench
scale, protease inhibitors and antioxidants are routinely added
to the extraction buffer to counter the denaturation,
degradation and structural modification by proteases
and phenolic compounds. Due to regulatory restraints and
low-cost requirement, these molecules should only be included
when deemed absolutely necessary for large-scale production.
Purification
Both chromatographic and non-chromatographic methods
have been employed to purify plant-derived pharmaceutical
proteins. Like proteins from other production hosts,
purification strategies are formulated for each individual
protein based on its solubility, size, pI, charge, hydrophobicity,
and affinity to specific ligands and the parallel characteristics
of host proteins. For mAb-based PMPs, while protein A or
G-based chromatography provides a superb and convenient
step (Langone, 1982), much effort is needed to eliminate plant
host molecules which cause resin fouling or/and interfere with
the binding of the target protein to protein A resin, and thus,
reduce its capacity. Non-chromatographic scalable processes
such as aqueous two-phase partitioning systems (ATPS) are
being developed to address this key issue (Platis and Labrou,
2006).
For other non-mAb-based vaccines and therapeutics, a
purification scheme has to be developed individually which
is based on multiple steps of conventional chromatographic
methods (Menkhaus et al., 2004). This time-consuming and
challenging process calls for the need to develop more
‘‘universal’’ or versatile purification methods. The employment
of affinity tags, particularly the tandem affinity tag purification
(TAP) strategy, provides possibilities for such versatile solution
PLANT VIRAL VECTORS
(Lichty et al., 2005; Arnau et al., 2006; Tagwerker et al., 2006).
However, the search for both efficient and precise proteases
for tag removal still presents serious challenges. In addition, the
inclusion of proteases in the manufacturing process further
complicates product purification, as well as raises cost and
regulatory concerns (Feeney et al., 2006; Kenig et al., 2006).
Several non-enzymatic affinity tag removal techniques have
been explored (Rais-Beghdadi et al., 1998; Wood et al., 1999),
but still require further optimization before becoming practical
for product manufacturing.
In addition to native proteins, carbohydrates and other host
molecules, unique plant endogenous molecules such as
phenolics needed to be removed during purification. Phenolics
tend to modify proteins by forming complexes, thus impeding
purification if not removed earlier during purification (Kusnadi
et al., 1998; Menkhaus et al., 2004). Some plants, such as
tobacco, also produce a high-level of toxic alkaloids that have to
be eliminated from final product (Twyman et al., 2003).
Regarding agroinfection, the potential elevated-level of
endotoxin (lipopolysaccharide, LPS) from a gram-negative
bacterium is another concern. Specific purification strategies to
lower the LPS to acceptable levels must be incorporated into
the overall scheme to ensure the safety of PMPs produced using
this platform (Magalhaes et al., 2007).
The overall purification design has to be robust, scalable,
cost-effective and compliant to cGMP regulations. Ideally, no
more than three chromatographic steps should be included to
obtain a highly purified product. Otherwise, recovery will
drop to an impractical level. Whenever possible, resins widely
accepted by standard pharmaceutical industry should be
considered first during process-development to minimize
future regulatory expenditure. The requirement for an
independent Quality Management System (QMS) to govern the
manufacture of pharmaceuticals also applies to the PMPs. In
addition to cGMP compliance during downstream processing,
analytical tests for releasing the final purified products have to
address plant specific contaminants in addition to the standard
required assays. Even though plants do not contain animal
viruses and other infectious agents, it must be validated
that LPS, phenolics, and alkaloids, as well as herbicides and
insecticides have been adequately removed from the final
product.
Conclusion
A significant increase in demand for high quality recombinant
proteins is already on the horizon. New biological systems for
the production of these proteins must be developed to meet
market demands. Plant expression systems based on viral
vectors have the greatest potential to provide such technology.
Once optimized and implemented at a commercial scale, these
expression systems should create a technology platform to
produce recombinant proteins with scalability, speed,
efficiency, cost-effectiveness and safety.
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