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Biotechnol. J. 2012, 7, 1099–1108
DOI 10.1002/biot.201100089
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Review
Using storage organelles for the accumulation
and encapsulation of recombinant proteins
Imran Khan1,2, Richard M. Twyman3, Elsa Arcalis1 and Eva Stoger1
1
Department for Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria
Institute of Molecular Biotechnology, RWTH Aachen, Aachen, Germany
3 Department of Biological Sciences, University of Warwick, Coventry, UK
2
Plants have been used to produce many diverse and valuable recombinant proteins, including subunit vaccines, antibodies and antibody fragments, hormones, blood products, cytokines, and enzymes. Different plant species and platforms have been explored as production hosts, each with
unique properties in terms of the gene transfer method, production time, environmental containment, scalability, downstream processing strategy, protein folding and accumulation, and overall
costs. Seed-based systems have many advantages because they exploit the natural storage properties of seeds, which facilitate batch processing and distribution. Seeds possess specialized storage organelles that may be used to accumulate recombinant proteins, offering stability both in
planta and after harvest in the final preparation/formulation. The post-harvest stabilizing effect of
seeds allows recombinant subunit vaccines and antibodies to be delivered via the mucosal route
because they are better able to withstand this harsh microenvironment when protected by the plant
matrix. Native storage organelles such as starch granules and protein bodies offer this protective
effect, but protein storage organelles can also be induced ectopically in vegetative tissues. In this
paper, we discuss the technical capabilities of storage organelle-based expression platforms and
their potential applications.
Received 12 NOV 2011
Revised 18 JAN 2012
Accepted 06 FEB 2012
Keywords: Biopharmaceuticals · Green biotechnology · Molecular farming · Seed-based production systems
1 Introduction
Molecular farming refers to the production of valuable recombinant proteins in plants and plant cells where the objective is to extract the product or use it in a purified form
or in partially processed plant tissue [1]. Plants provide
certain advantages over the more established platforms
based on microbes and mammalian cells because they are
inexpensive to grow, they are highly scalable and many
species have “generally regarded as safe” status. Plants
do not produce endotoxins (unlike bacteria) and they do
Correspondence: Prof. Eva Stoger, Department of Applied Genetics
and Cell Biology, University of Natural Resources and Life Sciences,
Muthgasse 18, 1190 Vienna, Austria
E-mail: [email protected]
Abbreviations: ELP, elastin-like polypeptides; PSV, protein storage vacuole;
PVC, prevacuolar compartment; TSP, total soluble protein
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
not support the replication of human viruses (unlike mammalian cells), but they do carry out eukaryotic post-translational modifications. These widely-cited advantages
prompted the development of molecular farming in the
1990s but were not sufficient to displace the well-established fermenter-based platforms, mainly because of early technical limitations in terms of overall yields, and the
absence of a mature regulatory framework for plant-derived pharmaceuticals [2]. More recently, several research
groups and industry-led consortia have developed
processes for the production of plant-derived pharmaceuticals that are fully compatible with good manufacturing
practice (GMP), and the first wave of drugs produced by
molecular farming is now approaching the clinic [3]. Many
different plant species have been considered as production hosts including leafy crops, cereal seeds, legume
seeds, oilseeds, fruits, vegetables, mosses, algae, and
aquatic plants [4]. In some cases, a variety of distinct platforms with its own advantages and disadvantages is
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available within a species, such as transgenic tobacco,
transplastomic tobacco, transient expression in tobacco
using Agrobacterium tumefaciens, plant viruses or hybrids thereof, hydroponic tobacco root secretion, hairy
root cultures, and tobacco suspension cells [5]. This diversity has resulted in a large number of independent molecular farming enterprises, some of which have been
more successful than others, but no real industry standard
platform equivalent to Escherichia coli in the bacterial
field and Chinese hamster ovary (CHO) cells among mammalian cell lines [4, 5].
Seed-based systems involve the targeted expression
of recombinant proteins in seeds and these are considered unique because of their very specific advantages
and applications [6–8]. The seeds of grain crops in particular have been developed as “bioreactors” for the production of a variety of recombinant proteins for medical and
industrial applications [9]. Cereal seeds are naturally
adapted for the storage of proteins in a stable and accessible form, a phenomenon that can be exploited to accumulate recombinant proteins [10]. Factors that influence
protein expression and stability in seeds include genotype selection, transgene copy number and zygosity, construct design, and protein targeting. Several publications
have shown convincingly that recombinant proteins expressed in mature seeds shown no detectable loss of activity at ambient temperature when stored for several
years [11, 12], even without specialized subcellular targeting [9]. Seed structure is advantageous for molecular
farming because seeds are small and homogenous in size,
thus concentrating the protein and facilitating downstream processing [6]. Seeds of various plant species have
been investigated and/or developed as platforms for molecular farming including rice [13], wheat [11], barley [14],
Arabidopsis [15], Brassica spp. [16], pea [17], safflower
[18], and soybean [19].
2 Seeds and their storage tissues
A seed is a small embryogenic plant that develops from an
ovule after fertilization. Each seed comprises three main
parts: the seed coat, endosperm, and an embryo with one
or two cotyledons. Structurally, plant seeds can be divided into three major categories: monocotyledonous seeds
(such as the cereals), endospermic dicotyledonous seeds
(such as tobacco), and non-endospermic dicotyledonous
seeds (such as beans). The embryo, which represents the
next plant generation, is enclosed by an endosperm that
provides nutrients. Both the embryo and the endosperm
are the product of a double fertilization process unique to
flowering plants. The seed coat provides a mechanical
barrier whose purpose is to protect the entire seed, including the embryo, enabling the plant to remain dormant. Different species have different storage priorities,
with some having evolved to store oils, others carbohy-
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drates (starch) and others proteins. Oil seeds store lipids
as triacylglycerol in spherical subcellular oil bodies. In
contrast, cereal endosperm cells predominantly contain
protein bodies (PBs) and starch grains [20].
Endospermic dicotyledonous seeds contain several
rows of endosperm cells surrounding the embryo. These
endosperm cells share the storage role with the cotyledon
cells of the embryo. In the case of non-endospermic seeds,
the endosperm is completely reabsorbed by the time seed
development is complete and the cotyledon cells store the
energy-rich compounds needed for germination. Monocotyledonous seeds such as cereals have a thin coat and
immediately underneath there are one or several rows of
aleurone cells depending on the species. The aleurone
cells divide to generate endosperm cells, and following
germination they release hydrolytic enzymes to make the
seed reserves available for the embryo. Adjacent to the
pedicel is a transfer layer also called “modified aleurone”
which is specialized for nutrient uptake and transport
from the maternal tissues to the developing endosperm
[21].
Cereal endosperm is a highly specialized tissue,
which often occupies most of the available space within
the seed, whereas the embryo is rather small in comparison. The endosperm is therefore the valuable food/feed
component of most cereal crops. Cereal seed endosperm
contains 70% starch by dry weight, comprising the α-glucan polymers amylose and amylopectin packed into semicrystalline granules in amyloplasts [22]. In maize, starches begin to accumulate 10 days after pollination (DAP)
whereas in other cereals the accumulation of starch granules in endosperm cells begins soon after cellularization
[20]. Cereal endosperm is also the major sink for storage
proteins, and these contribute processing properties such
as dough elasticity, which in the case of wheat is essential for making bread and pasta [23].
Most of the different seed types and storage tissues
have been successfully developed for the production and
accumulation of recombinant proteins, and some examples are listed in Table 1.
3 Targeting proteins to storage organelles
The subcellular localization of a recombinant protein
strongly influences its stability, accumulation, and ultimately the yield on recovery, but there is no ideal strategy because proteins have diverse structures and properties. The wealth of data in the literature provides guidance in terms of good strategies for particular types of proteins, e.g., there have been many reports of recombinant
antibodies expressed in plants, and because their stability and functionality rely on polypeptide folding, multimer
assembly, the formation of disulfide bonds and glycosylation, targeting to the secretory pathway is recommended.
This is achieved by adding an N-terminal signal peptide
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Table 1. Examples of recombinant proteins produced in seeds
Crop
Recombinant protein
Plant tissue/
subcellular localization
Expression level/yield Promoter
Arabidopsis
A. thaliana
Human α-L-iduronidase
(IDUA)
Endoplasmic reticulum
(ER)
18 μg IDUA/mg TSP
Reference
Regulatory sequence Phaseolus
vulgaris arcelin 5-I gene
[15]
Arabidopsis oleosin promoter
[29]
Hirudin
Oil-bodies
1% TSP
Soy β-coglycinin α-subunit
Promoter
[78]
Spider dragline silk
protein DP1B
Endoplasmic reticulum
(ER) and vacuole
18% TSP in ER
8.2% TSP in Vac
β-Phaseolin promoter
[79]
Single-chain variable
fragment (scFv)-Fc
antibodies
Endoplasmic reticulum
(ER)-derived
compartments
19–28 μg/mg of seed
Barley H. vulgare
HIV Diagnostic Reagent
Endoplasmic reticulum
150 μg of reagent/g
B × 17 promoter
[80]
Maize Z-mays
HIV 2G12
Endosperm/embryo
38–75 μg per gram
of dry seed weight
Endosperm-specific rice
[81]
HIV 2G12SEKDEL
ER-derived protein Bodies 5.7% TSP
Glutelin-1 (gt-1) promoter
[52]
Avidin
Endoplasmic reticulum
(ER)
Maize ubiquitin promoter
[82]
27-kDa γ-zein promoter
[83]
[17]
1.3 g/kg
Escherichia coli heat
Starch granules
labile enterotoxin (LT-B)
Pea P. sativum
Safflower
C. tinctorius
Soybean G. max
Anti-Eimeria scFv
antibody fragments
Seed
1.76 mg per 1 g dry
seed weight
Seed specific USP promoter
Human serum albumin
(HSA)
Endosperm
2.75 g/kg of brown
rice
Endosperm-specific promoter, [84]
Gt13a
7Crp peptide
ER-derived protein bodies 60 μg/grain
Glutelin GluB-1
[53]
Human interleukin
Endoplasmic reticulum
4.5 mg/100 g
Glutelin GluB-1
[85]
Cholera toxin B subunit
(CTB)
Protien bodies (PB)
30 μg of CTB per seed Glutelin GluB-1
[12]
Aspergillus niger phytase Protein storage vacuoles
(PSVs) and the protein
bodies
0.5% TSP
Gt1 promoter
[86]
Apolipoprotein AI Milano Apoplast
(ApoAIMilano)
7.3 g/kg seeds
Phaseolin promoter
[18]
Human insulin
Oilbodies or ER
Enterotoxigenic
ER-derived protein bodies 2.4% TSP
Soybean glycinin promoter
[87]
[9]
Human basic fibroblast
growth factor (bFGF)
Seed
2.3% TSP
Glycinin G1
[88]
Human coagulation
factor IX (hFIX)
Protein storage vacuole
(PSV)
0.8 g/kg seed
α-Subunit of the β-conglycinin
promoter
[19]
Human acid
β-glucosidase (GCase)
Protein storage vacuoles
(PSV)
3% TSP
Soybean 7S globulin promoter
[89]
Single-chain Fv antibody Proposed. vacuole and
fragment (scFv)
apoplastic fluids
0.2% TSP
35S cauliflower mosaic virus
promoter
[90]
Anti-HIV-1 antibody
2G12
0.3% TSP
CaMV 35S promoter
[70]
Ubiquitin-1 promoter
[51]
Ubiquitin-1 promoter
[11]
E. coli heat-labile toxin B
subunit (LTB)
Tobacco
N. tabacum
Wheat T. aestivum Human serum albumin
Protein storage vacuole
(PSV)
ER-derived protein bodies 0.5% TSP
Single-chain Fv antibody Endosperm
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
30 μg/g
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to each antibody chain, which usually leads to the protein
being secreted to the apoplast, although an optional Cterminal KDEL or HDEL tetrapeptide will retrieve secreted proteins to the endoplasmic reticulum (ER) [24].
The precise yields of a recombinant antibody
achieved with this strategy vary from case to case because there are intrinsic differences which also affect stability, but one ER-targeted antibody accumulated to nearly 7% total soluble protein (TSP) in transgenic tobacco
[25], probably reflecting the efficient folding of the
polypeptides by ER molecular chaperons, the favorable
environment for disulfide bond formation and the absence
of significant protease activity. Proteins without a signal
peptide generally accumulate in the cytosol and this is a
poor environment for most antibodies, resulting in yields
typically below 0.1% TSP [24].
Protein targeting also plays a key role in many types of
post-translational modifications, among which glycosylation is the most relevant for molecular farming because
the glycan chains can affect protein structure, biological
function and immunogenicity. Targeting to a specific
compartment may therefore influence the interrelated
processes of folding, assembly and ultimate accumulation
site (deposition), which have a direct impact on protein
stability and yield [1]. Targeting may also achieve specific post-translational modifications, such as particular Nglycan structures, because glycans added in the ER undergo further modification in the Golgi apparatus [5, 8].
Although the description above applies generally to
plant cells, seed storage tissues are highly specialized and
often have a larger, more complex endomembrane system
than vegetative cells through which storage proteins travel to reach their final destination. The endomembrane
system comprises an abundant ER reminiscent of secretory cells in animals, ER-derived PBs and different types
of vacuoles. Seeds therefore offer several alternative subcellular destinations for recombinant proteins including
PBs derived from the ER, protein storage vacuoles (PSVs),
starch granules and the surface of oil bodies. All of these
destinations have been tested to see if they increase recombinant protein stability, enhance accumulation, facilitate recovery and purification, or provide additional benefits such as increasing the efficacy of oral vaccine delivery.
3.1 Oil bodies
Lipid particles are found in the seeds, flowers, pollen
grains, fruits, and stems of higher plants, but they are especially prevalent in the seeds of “oilcrops” such as sunflower, safflower, rapeseed, and mustard. In these seeds,
cells contain abundant spherical oil bodies approximately 0.5–1 μm in diameter, comprising a triacylglycerol matrix surrounded by a layer of phospholipids. Oil bodies are
relatively stable because they do not aggregate or coalesce and they are surrounded by a protective layer of
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15–26 kDa proteins known as oleosins, which provide a
recognition signal for lipase binding during oil mobilization in seedlings [26]. In maturing seeds, the triacylglycerols, phospholipids, and oleosins are synthesized in the
ER, which releases oil bodies [27].
Protein targeting to oil bodies has been achieved by
expressing recombinant proteins as oleosin fusions,
which causes them to accumulate at the periphery of the
oil bodies [27]. This co-accumulation strategy not only increases recombinant protein stability by sequestering the
proteins in a favorable environment, it also facilitates purification because the recombinant protein is enriched in
the oil body fraction, which is easy to separate. The recombinant protein can then be released from its fusion
partner by endoprotease digestion. Using this approach,
it has been possible to express the pharmaceutical protein hirudin in Brassica napus [28] and Brassica carinata
[16] and recombinant apolipoprotein A1 Milano in safflower seeds (Carthamus tinctorius), the latter at a level
equivalent to 7 g of recombinant protein per kg of seed
[18]. One of the closest molecular farming products to the
market is recombinant human insulin produced in safflower seeds by the Canadian biotechnology company
SemBioSys Inc., which is currently undergoing phase III
clinical trials [3].
The main advantage of oil body targeting is the relatively low cost of processing brought about by the convenient purification strategy, but this is not always a trouble-free process. Several reports describe incomplete separation of recombinant proteins from oleosin [29] and the
need for post-recovery proteolytic cleavage can reduce
the overall recovery as well as increasing costs [28–30].
Therefore, it remains to be seen whether this technology
will be adopted widely or will remain as a niche platform.
3.2 Starch granules
Starch is the primary storage polysaccharide in all plants,
and comprises a mixture of linear amylose and highlybranched amylopectin, both of which are polymers of glucose [31]. Starch is transiently deposited in chloroplasts
(transitory starch) but accumulates as semi-crystalline
granules (1–100 μm in diameter and in a variety of shapes)
in the specialized amyloplasts of storage organs [31]. In
cereals such as wheat and barley, amyloplasts contain a
mixture of small and large starch granules known as A and
B granules, which differ in morphology and chemical
composition [32, 33]. In wheat for example, A-granules are
disks 10–38 μm in diameter whereas B-granules are
spheres or polygons <10 μm in diameter [34].
Enzymes that break down starch possess highly conserved and mostly C-terminal starch-binding domains
(SBDs) approximately 100 amino acids in length. These
are conserved in three different families of glycosidases
despite their different catalytic mechanisms [35–37] and
are therefore found in bacterial and fungal α-amylases,
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bacterial cyclodextrin glucanotransferases, bacterial βamylases and fungal glucoamylases [38]. SBDs retain their
function when fused to other proteins and can therefore
be used to target recombinant proteins to existing and
nascent starch granules [38, 39].
3.3 Protein bodies and protein storage vacuoles
Although dicotyledonous seeds store proteins predominantly in PSVs, cereal endosperm contains two types of
protein storage organelles: PBs, which are derived from
the ER, and PSVs, which are formed de novo [23, 40, 41].
The storage organelles contain different classes of storage
proteins. PSVs are the main target sites for the accumulation of globulins and albumins whereas the PBs contain
prolamins, which are predominantly found in cereal
species [23, 41]. PBs generally form directly within the lumen of the rough ER. If they reach a sufficient size, they
may bud from the ER as discrete spherical organelles and
remain as such in the cytosol, or they may be sequestered
into PSVs by autophagy, as shown in wheat, barley and
oat [42, 43]. The formation of PBs is complex and the exact mechanism is unclear. PSVs contain three morphologically distinct regions: the matrix, crystalloid and
globoid [40]. This coincides with the presence of integral
membrane proteins in the PSV crystalloid, strongly arguing for the presence of a distinct storage compartment
within PSVs [44].
The trafficking of native proteins to PBs and the PSV
is convoluted and differs among storage protein types and
plant species (Fig. 1) which means targeting recombinant
proteins to specific storage organelles can be similarly dif-
ficult to control. The trafficking of endogenous seed storage proteins is therefore particularly relevant in the context of molecular farming because individual proteins follow specific and idiosyncratic routes through the endomembrane system to PBs or the PSV, in some cases
passing through the Golgi apparatus and in other cases
bypassing this compartment [23, 40, 42, 45]. For example,
albumins and globulins in most plant species are transported from the ER lumen to PSVs through the Golgi apparatus via dense vesicles [46], but in pumpkin seeds
these proteins bypass the Golgi apparatus and reach the
PSV via precursor-accumulating (PAC) vesicles [47].
The trafficking of storage proteins in cereals has been
studied extensively because seed proteins contribute significantly to the nutritional value of cereal grains. Cereal
globulins follow the pathway described above, accumulating in the PSVs after passing through the Golgi apparatus. In contrast, prolamins follow different pathways in
different species. In rice, maize, and sorghum, prolamins
aggregate into dense PBs within the lumen of the rough
ER where they remain protected from degradation and
desiccation until they are required by the germinating
seedling [48]. This isolation and protection makes PBs
particularly useful for the accumulation of recombinant
proteins that might be sensitive to proteolysis if secreted,
and retention within ER-derived compartments eliminates the potential for complex end-point plant-type glycosylation [49]. In wheat and oat, prolamins accumulate to
form aggregates and bud from the ER as in other cereals,
but here they are later absorbed by PSVs through an autophagy-like process, bypassing the Golgi apparatus [42,
50].
Figure 1. Schematic overview of protein
trafficking pathways to storage organelles in seeds. ER-derived PBs and
protein storage vacuoles are the main
target sites for the accumulation of
endogenous and recombinant proteins.
These organelles may be reached via
diverse pathways in the embryonic and
endosperm tissues of different plant
species. TGN trans-Golgi network, PAC
precursor-accumulating vesicles, MVB
multi-vesicular bodies, PVC pre-vacuolar
compartment.
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Although these data suggest that the fate of a recombinant protein expressed in seeds may be unpredictable
as well as species-dependent [51], PBs and the PSV have
each been used as targets for the accumulation of recombinant proteins in seeds by exploiting the KDEL retrieval
signal or sequence motifs that promote protein aggregation [13, 51–54]. There is also evidence that mRNA targeting may play a crucial role in the deposition of prolamins in rice endosperm, since prolamins and globulins/glutelins are synthesized on two separate sub-domains of the ER. Prolamin mRNAs are targeted to the ER
membrane surrounding PBs by an mRNA signal recognition process, whereas globulin/glutelin mRNA is mainly
directed to ribosomes on the cisternal ER [55, 56]. Targeting strategies based on mRNA sequences may therefore
provide alternative strategies for controlling recombinant
protein accumulation in cereal seeds.
4 Induced protein-storage organelles
The accumulation of recombinant proteins can be enhanced by expressing them as fusions with partner proteins that improve stability. This has been achieved, e.g.,
by expressing proteins as fusions with the cholera toxin B
subunit [57], viral coat proteins [58], ubiquitin [59], β-glucuronidase [60], and human immunoglobulin [61]. The
use of seed storage proteins as stabilizing partners provides additional advantages because PB-like aggregates
can be induced in plant tissues that normally lack such organelles, offering further advantages such as protection
from degradation, alternative purification strategies, and
post-harvest encapsulation. Under these circumstances,
storage organelles can form even in non-seed tissues,
thus combining the advantages of seed-based systems
with the rapid biomass accumulation that is possible in
leafy plants. Protein fusion partners that provide these enhanced benefits include natural zeins, synthetic elastinlike polypeptides (ELP), and fungal hydrophobins (Table
2), all three of which can induce PBs and facilitate the purification of recombinant proteins even at process scale
[62].
4.1 Zein fusions
Zeins are the major storage proteins (prolamins) in maize
endosperm where they accumulate in the ER and bud off
as PBs. They do not contain the canonical HDEL/KDEL retrieval signals characteristic of other ER luminal proteins,
which suggests they have an intrinsic retention capability that may be transferrable to other (recombinant) proteins, although the mechanism is not understood. Experiments with mutated γ-zein sequences in Arabidopsis
have shown that the proline-rich N-terminal domain
(which contains eight PPPVHL repeats) and the cysteinerich C-terminal domain are required to trigger zein body
formation [63].
The N-terminal sequence of γ-zein has also been
shown to self-assemble and induce ER-derived PBs in tobacco, Arabidopsis, and rice, and remarkably also in insect cells (Spodoptera frugiperda), mammalian CHO and
COS cells, and filamentous fungi (Trichoderma reesei) [49,
62]. This sequence has been fused to several recombinant
proteins, which were subsequently incorporated into PBs
thereby increasing the overall yield [64–66].
The γ-zein domain has also been fused to the bean
storage globulin phaseolin, and the recombinant fusion
protein (zeolin) subsequently accumulated to levels in excess of 3.5% TSP [64]. Although the γ-zein domain was unable to increase the accumulation of HIV-1 Nef protein either as an N-terminal or C-terminal fusion, the expression
of a fusion protein comprising HIV-1 Nef and the entire recombinant zeolin resulted in expression levels of up to
1.5% TSP in transgenic tobacco plants and the formation
of ER-derived PBs [67].
4.2 Elastin-like polypeptides
ELPs are synthetic biopolymers comprising repeats of the
peptide sequence Val-Pro-Gly-Xaa-Gly, where Xaa is any
amino acid except proline [68]. ELPs have two remarkable
properties. First, proteins fused to ELPs form PBs of similar size and morphology to natural prolamin bodies, as
demonstrated using an ELP fusion to green fluorescent
protein (GFP) which significantly increased the stability
and accumulation of the recombinant protein in tobacco
leaves [62]. Second, the structure and solubility of ELPs is
Table 2. Fusion partners for the creation of artificial storage organelles. Reviewed in detail by [62]
Fusion sequence
Origin
Purification method
Increased yield
ER signal
Zein
Plant
Isopycnic sucrose
density centrifugation
15–100 fold
Protein repeat domain, Pro-X domain, cysteine domain
Elastin-like
polypeptides
Animal
Inverse transition cycling
2–100 fold
K/HDEL sequence
VPGXG
Hydrophobin
Fungus
Surfactant-based aqueous
two phase system
Unknown
K/HDEL sequence
Hydrophobic interaction,
eight cysteine residues
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PB formation
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temperature-dependent, which means ELP fusion proteins can be purified using a simple and inexpensive
(non-chromatographic) method called inverse transition
cycling that involves reversible precipitation. ELPs are
converted to hydrophobic aggregates and form β-spiral
structures when heated to the transition temperature,
and this thermal response is maintained in ELP fusions,
allowing recombinant proteins to be recovered by inverse
transition cycling [62]. Heat sensitivity is directly related
to the size of the fusion protein, such as that the transition
temperature could be 30–39 °C for proteins ranging from
30–65 kDa. ELP fusions are up to 40 times more abundant
than control proteins lacking the ELP when expressed in
tobacco seeds [69] although their impact on protein quality remains to be determined [70].
4.3 Hydrophobins
Hydrophobins are small (∼10 kDa) and strongly hydrophobic fungal proteins that were initially developed as fusion
tags to facilitate the purification of recombinant proteins
from the supernatant of fungal cultures by surfactantbased aqueous two-phase partitioning [62–72]. This is a
simple, rapid, and inexpensive method that allows the purification of recombinant proteins from large culture volumes [73] but it has also been shown to increase the stability and accumulation of recombinant proteins in fungi
and also in plants [71].
Recombinant hydrophobin fusion proteins targeted to
the ER induce the formation of novel PBs in fungi [62] and
in tobacco leaves [74]. Recently the hydrophobin HFBI sequence from Trichoderma reesei was fused to GFP and introduced into tobacco leaves, resulting in an unprecedented expression level equivalent to 51% TSP [74].
5 The advantage of bioencapsulation applied
to the production of vaccine candidates
Because many pathogens enter the body via mucosal surfaces, both passive and active immunization strategies
targeted to the mucosa have been developed with many
researchers focusing on oral delivery [75]. The production
of vaccine antigens in plants therefore has clear benefits,
because plant material containing vaccine antigens can
be consumed directly, avoiding the need for expensive
processing and purification. However, one of the drawbacks of oral immunization is the potential destruction of
vaccine antigens in the digestive system before they can
reach the immune cells in the ileum, which are clustered
in regions known as Peyer’s patches. In this context, the
production of vaccine antigens in plant tissues provides a
further advantage because the plant matrix provides limited protection from digestive enzymes and allows more
time for the antigen to interact with immune cells. This
protective effect is enhanced by the incorporation of re-
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
combinant proteins into storage organelles such as starch
granules [76] and PBs [54].
The study by Takagi et al. [54] is particularly noteworthy because they compared the survival of a known oral
tolerogen when administered as a synthetic peptide or as
recombinant rice grains in which the antigen was targeted either to ER-derived PBs or PSVs. Both the endospermderived peptides were more resistant to in vitro digestion
with pepsin than the soluble form, but feeding studies indicated that PBs were more effective and therefore appeared to provide greater protection than PSVs. These
data suggested that oral delivery of the bioencapsulated
form of the tolerogen significantly enhanced its immunological efficacy for the suppression of allergen-specific IgE
responses. This agrees with earlier studies showing that
rice prolamin bodies are only digested to a limited extent
and many prolamin bodies are excreted [77]. Similar results have been presented for the cholera toxin B subunit
[12] and a recombinant antibody expressed in pea seeds
[17]. In the latter case, flour from the transgenic pea seeds
was more potent than the purified antibody fragments,
and additional experiments showed that the antibody
was protected from degradation in the pea seeds, possibly reflecting the presence of protease inhibitors [17].
6 Conclusions
Seed-based systems for molecular farming exploit natural
storage properties, which include the use of storage organelles to accumulate recombinant proteins in a stable
and accessible form. Recent investigations of the trafficking of endogenous seed storage proteins have shown that
multiple pathways are involved and that these rely on a
combination of well-characterized signals based on short
peptide sequences, and intrinsic properties of storage
proteins whose exact principles have yet to be established. Although there is some uncertainty as to how storage proteins are directed through the membrane system,
it appears that their properties are often cis-dominant
such that fusion proteins comprising a valuable target
and a storage protein not only accumulate to higher levels than the target protein alone, but in many cases induce the formation of ectopic storage compartments even
in non-seed tissues thus conferring all the advantages of
the seed-based expression platform. One of these advantages is bioencapsulation, which means that recombinant subunit vaccines and antibodies can be delivered
via the mucosal route because the plant matrix protects
them at least temporarily from the harsh mucosal microenvironment. It is therefore likely that seed-based expression platforms will play a play a prominent role in the
development of future oral vaccine candidates.
The authors declare no conflict of interest.
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Eva Stoger is a Professor of Molecular
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1106
Plant Physiology at the Department of
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