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Cell Biology International ISSN 1065-6995

doi: 10.1002/cbin.10304

REVIEW

Plant-based biopharming of recombinant human lactoferrin


Alla I. Yemets, Iryna V. Tanasienko, Yuliya A. Krasylenko and Yaroslav B. Blume*
Department of Genomics and Molecular Biotechnology, Institute of Food Biotechnology and Genomics, National Academy of Sciences of Ukraine,
Osipovskogo Str., 2a, Kyiv 04123, Ukraine

Abstract
Recombinant proteins are currently recognized as pharmaceuticals, enzymes, food constituents, nutritional additives,
antibodies and other valuable products for industry, healthcare, research, and everyday life. Lactoferrin (Lf), one of the
promising human milk proteins, occupies the expanding biotechnological food market niche due to its important versatile
properties. Lf shows antiviral, antimicrobial, antiprotozoal and antioxidant activities, modulates cell growth rate, binds
glycosaminoglycans and lipopolysaccharides, and also inputs into the innate/specic immune responses. Development of
highly efcient human recombinant Lf expression systems employing yeasts, lamentous fungi and undoubtedly higher
plants as bioreactors for the large-scale Lf production is a biotechnological challenge. This review highlights the advantages
and disadvantages of the existing non-animal Lf expression systems from the standpoint of protein yield and its biological
activity. Special emphasis is put on the benets of monocot plant system for Lf expression and the biosafety aspects of the
transgenic Lf-expressing plants.
Keywords: human lactoferrin; recombinant expression systems; plant-based biofarming; plant-pathogen resistance;
biosafety

Introduction
Recently numerous recombinant proteins have been used
intensely in pharmacy, industry and research and, therefore,
have to meet a range of sophisticated quality requirements,
before they could be considered safe, in particular, an
extra high-purity (Ma et al., 2003). According to Good
Manufacturing Practice, all recombinant proteins must be
sufciently pure and homogeneous with contaminants
removed to acceptable levels (Fischer et al., 2012). It is
important either to improve the protein production from
their native sources or to search for new ones together with
the development of the efcient protein expression systems
and the advance of protein extraction protocols.
Novel recombinant proteins, also referred to as highmolecular drugs, could be the targeted agents for the
treatment of such common health problems of industrial
countries as oncological, cardiovascular and infectious
diseasesall critical to an expanding and aging human
population (Elbehri, 2005). The existing pharmaceutical

industry is based on the chemical synthesis and/or


production of the organic molecules by transgenic microorganisms, mammalian cell cultures, or animals (Schwartz,
2001; Ma et al., 2003). However, the eukaryotic folding of the
recombinant proteins and their proper posttranslational
modications (e.g., glycosylation and phosphorylation)
are the basic prerequisites for the protein biological activity
that cannot be ensured into the transgenic prokaryotes
(Houdebaine, 2000; Ma et al., 2003). Mammalian cell
cultures and transgenic animals also have such numerous
disadvantages as time-consuming protein expression,
unprotable purication procedure and, additionally, the
risk of contamination with viral and oncogenic DNAs
(van Berkel et al. 2002; Stefanova et al., 2008). The promising
trend in biotechnology is the use of plants as green
bioreactors for recombinant protein production. At the
same time, the key advantages of plant expression
systems are sufcient protein yield, eukaryotic protein
folding along with comparatively short life cycles, easy seed
storage, absence of animal, and human viruses, and, last

Corresponding author: e-mail: cellbio@cellbio.freenet.viaduk.net

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Plant-based biopharming of recombinant human Lf

A.I. Yemets et al.

but not least, the low-cost protein production (Elbehri,


2005).
Characteristics of the key functions of lactoferrin
Human lactoferrin (Lf) is considered to be one of the
conventional rst range proteins for biopharming due to
its numerous biological activities (Stefanova et al., 2008). Lf,
a 80 kDa globular protein from the transferrins family, is
produced by mucosal gland cells of various mammalian
species, and can be found in all secretory uids (milk,
colostrum, saliva, tears, etc.) (Adlerova et al., 2008; GonzlezChvez et al., 2009; Lnnerdal and Suzuki, 2013). Apart from
its main physiological functions, namely binding and
transport of iron ions, Lf exhibits antiviral, antimicrobial,
antiprotozoal and antioxidant activities. It is also able to
modulate cell growth rate and to bind viral glycosaminoglycans and bacterial lipopolysaccharides (Wakabayashi et al.,
2006). Pepsin digestion of Lf generates its fragment
lactoferricin (Bellamy et al., 1992) that also reveals potent
bactericidal activity against antibiotic-resistant strains of
Staphylococcus aureus and Escherichia coli from clinical
origins (Flores-Villase~
nor et al., 2010). Moreover, recombinant Lf has been tested for clinical use in treatment and
prevention of such human and animal diseases as solid
tumors (Hayes et al., 2005, 2010) and diarrhea (Humphrey
et al., 2002). Its use as vaccine adjuvant modulates the
adaptive immune response in humans (Hwang et al., 2011).
Lf is an iron-binding protein, and its functions can be
related to this property (Lnnerdal and Suzuki, 2013). Thus,
one of the mechanisms of Lf antimicrobial properties is
based on its ability to sequester iron ions from the bacterial
pathogens (Garca-Montoya et al., 2012). Lf can eliminate
microorganisms via iron-independent pathway (Valenti and
Antonini, 2005) by the direct interaction with the bacterial
cell surface (Bortner et al., 1989; Farnaud and Evans, 2005;
Garca-Montoya et al., 2012) and release lipopolysaccharide
(LPS) from the cell wall of Gram-negative bacteria causing
poration that allows exposure of the inner membrane
proteoglycan layer to lysozyme activity (Ellison and
Giehl, 1991; Lnnerdal and Suzuki, 2013).
Lf has antiviral activity against a broad range of RNA and
DNA-containing human and animal viruses (GarcaMontoya et al., 2012; Lnnerdal and Suzuki, 2013) by
inhibiting virushost interaction, virus trafcking or direct
binding of the viral particle by the blocking of glycosaminoglycan viral receptors, especially heparan sulfate (GarcaMontoya et al., 2012). It acts strongly against HIV in vitro
(Garca-Montoya et al., 2012) by inhibiting viral replication
inside the host cell (Swart et al., 1996; Qiu et al., 1998;
Garca-Montoya et al., 2012). Moreover, the interaction of Lf
with nucleolin surface blocks the attachment and entry of
HIV particles into HeLa P4 cells (Legrand et al., 2004).
990

Lf also possesses antifungal activity (Gifford et al., 2005)


by its direct interplay with the pathogen and Fe3
sequestration (Zarember et al., 2007; Gonzlez-Chvez
et al., 2009). Thus, Lf eliminates Candida albicans and
C. krusei by the alterating the permeability of their cell
surfaces (Wakabayashi et al., 1996; Garca-Montoya et al.,
2012).
Many reports indicate the benecial effects of bovine and
human Lf in cancer treatment (Gibbons et al., 2011;
Vogel, 2012), including chemically induced tumors in
laboratory rodents (Adlerova et al., 2008). Lf prevents cell
cycle transitions from G1 to S (Damiens et al., 1999) and G0
to G1 phases (Xiao et al., 2004), and modulates cytokine
production in malignant cells (Garca-Montoya et al., 2012).
It can promote apoptosis and arrest tumor growth in vitro
(Lnnerdal and Suzuki, 2013). Among the other factors
associated with Lfs anticancer effects are the downregulation of phase I detoxifying enzyme and cytochrome
P450 1A2 (Fujita et al., 2002), and the upregulation of phase
II detoxifying enzyme and glutathione-S-transferase, with a
consequent decrease in carcinogen activation (Tanaka
et al., 2000).
Fluctuation in Lf content may be also used as biomarker
for disease indication (Vogel, 2012), such as chronic
periodontitis (Glimvall et al., 2012), ulcerative colitis and
Crohns disease (Vogel, 2012). Though Lf levels are
increased in the synovial uid of the inamed knee joints,
suggesting neutrophil inltration, the Lf levels in serum
were indistinguishable from healthy controls (Caccavo
et al., 1999).
Lf can affect wound healing both in vitro and in vivo
(Fujihara et al., 2000; Lyons et al., 2007; Pattamatta et al.,
2009). Its concentration is associated with free fatty acid
content after fat overload (Fernandez-Real et al., 2010;
Lnnerdal and Suzuki, 2013), suggesting an important
role of Lf in fat metabolism due to its antiadipogenic,
antioxidative and anti-inammatory activities (Lnnerdal
and Suzuki, 2013).
Despite numerous benecial effects of this multifunctional protein in the treatment of various infectious diseases,
little is understood about its mechanisms of action.
Comparative characteristics of the existing
eukaryotic non-animal systems of lactoferrin
expression
Recombinant Lf (rLf) has been expressed in a range of
organisms including bacteria: Escherichia coli (Tian et al.,
2007) and Rhodococcus erythropolis (Kim et al., 2006); yeasts:
Pichia pastoris (Kruzel and Zimecki, 2002; Jiang et al., 2008;
Jo et al., 2011) and Saccharomyces cerevisiae (Liang and
Richardson, 1993; Conneely et al., 2001); lamentous fungi:
Aspergillus oryzae, A. nidulans (Conneely et al., 2001; Ward

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A.I. Yemets et al.

et al., 1992a) and A. awamori (Ward et al., 1995); higher


plants: tobacco (Nicotiana tabacum L.) (Salmon et al., 1998)
and Australian tobacco (N. bentamiana Domin.) (Li
et al., 2004), potato (Solanum tuberosum L.) (Chong and
Langridge, 2000), tomato (Lycopersicon esculentum Mill.)
(Lee et al., 2002), pear (Pyrus sp.) (Malnoy et al., 2006), rice
(Oryza sativa L.) (Nandi et al., 2002), ginseng (Panax ginseng
C.A. Meyer) (Kwon et al., 2003), sweet potato (Ipomoea
batatans (L.) Lam.) (Min et al., 2006), eleuthero (Acanthopanax senticosus (Rupr. & Maxim) Harms) (Jo et al., 2006),
Thale cress (Arabidopsis thaliana (L.) Heynh.) (Nguyen
et al., 2011), barley (Hordeum vulgare L.) (Tanasienko
et al., 2011), wheat (Triticum aestivum L.) (Han et al., 2012)
and alfalfa (Medicago sativa L.) (Stefanova et al., 2013),
insects fall armyworm (Spodoptera frugiperda Smith.)
(Zhang et al., 1998a) and silkworm (Bombyx mori L.) (Liu
et al., 2005); mammals: cow (Bos sp. Bojan.) (van Berkel
et al., 2002) and goat (Capra sp. L.) (Han et al., 2007). In spite
of the large list of rLf expressing systems, only a few have
been approved for the market and/or introduced in clinical
practice. For instance, Aspergillus niger-produced Lf (trade
name talactoferrin) (Ward et al., 1995) is used for solid
tumors treatment (Hayes et al., 2005, 2010) and Oryza
sativa-derived Lf (trade name lacromin) is an antiapoptotic cell culture media supplement that increases cell
growth rate (InVitria, Ventria Bioscience, USA, http://www.
invitria.com). In this review, the advantages and disadvantages of yeast, fungal, and plant expression systems for
recombinant human Lf production (rhLf) are discussed
touching upon Lf biosafety aspects. The up-to-date approach
concerning the use of Lf plant expression systems for the
enhancement of non-specic plant pathogen resistance is
also highlighted.
Such mammalian expression systems as cow (van Berkel
et al., 2002) and goat (Han et al., 2007) have numerous
disadvantages, including potential risk of viral contamination of target proteins, long life cycle, expensive purication
procedures, as well as bioethical concerns (Stefanova
et al., 2008). Therefore, new Lf sources and/or biofactories
enabling high expression levels of target protein for its largescale production, easy extraction and purication procedures are required. For this reason, such widely used
producers as yeast, lamentous fungi, and higher plants are
being considered as the most efcient eukaryotic systems for
Lf expression.
Yeast have been used since ancient times as universal
bioreactors suitable for pharmaceutical and nutrient protein
production because of their eukaryotic protein folding
system, simplicity of cultivation and common protocols for
gene manipulations in the majority of unicellular organisms
(Cereghino and Cregg, 1999). The range of the advantages,
such as target gene expression under the strong regulated
alcohol oxidase I (AOX1) promoter, marker/host strain

Plant-based biopharming of recombinant human Lf

combination and also the high cell culture density, make


P. pastoris an efcient bioreactor for recombinant human Lf
production (Cereghino and Cregg, 1999; Kruzel and
Zimecki, 2002; Jo et al., 2011). In order to increase hLf
expression in P. pastoris, a codon-optimized hLf gene was
fused to 11 different signal sequences for the identication of
the optimal one that facilitates the translocation of rhLf into
the secretory pathway and nally into the culture medium
(Choi et al., 2008). From all tested S. cerevesiae signal
sequences, alpha mating factor prepro has been selected
and modied to facilitate the processing of the protein
prior to mature rhLf secretion. The resulting sequence
(ScaMFppKR) led not only to the facilitation of hLf
secretion, but it also minimized its intracellular accumulation. Two promoters widely used in yeast inducible alcohol
oxidase 1 (pAOX1) (Cereghino and Cregg, 1999, 2000) and
constitutive P. pastoris glyceraldehyde-3-phosphate dehydrogenase (PpGAPDH) were also tested for their ability to
express rhLf. The combination of AOX1 promoter, codonoptimized hLf gene and ScaMFppKR signal sequence from
S. cerevesiae allowed 99.8 mg/L of Lf to be reached (Choi
et al., 2008). Moreover, the microbial surface display
approach has been proposed to increase hLf level based
on its expression in P. pastoris, with the consequent
immobilization of the protein on the cell surface (Jo
et al., 2011). The hLf gene was fused to glycosylphosphatidylinositol (GPI)-anchored protein of S. cerevisiae as an
anchoring motif and expressed under the control of the
AOX1 promoter. Analysis of this expression system
conrmed the localization of rhLf in P. pastoris membrane
as an integral protein closely associated with other cellular
proteins (Jo et al., 2011). Lf produced in P. pastoris does not
undergo core-fucosylation of N-linked glycans typical of
human neutrophilic leukocytes, whereas human milkderived Lf displays fucose residues on N-acetylglucosamine
(Choi et al., 2008).
In spite of ancient biotechnological traditions and evident
progress in genetic and molecular biology, the use of S.
cerevisiae as Lf expression system has some limitations
concerning its lower secretory capacity compared with other
yeasts species (Cereghino and Cregg, 1999). Nevertheless,
the optimization of the vector construction via invertase
signal sequence enhanced the hLf yield in S. cerevisiae to
1.52 mg/L (Liang and Richardson, 1993). However, the
signicant disadvantage of yeasts as heterologous protein
bioreactors is their inability to provide proper eukaryotic
protein post-translational modications as amidation and
prolylhydroxylation, and also some types of phosphorylation and glycosylation (Cregg and Higgins, 1995).
In turn, the secretion potential of lamentous fungi
distinguishes these organisms favorably from the other
perspective producers of bioactive proteins. Filamentous
fungi, in contrast to prokaryotes, can provide proper

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eukaryotic post-translational modications of polypeptides


as N-glycosylation and disulde bonds formation (Maras
et al., 1999). Lf expression in three lamentous fungi:
A. oryzae under the control of a-amylase promoter and the
30 -anking region of the A. niger glucoamylase gene, and in
A. nidulans under the control of strong ethanol-inducible
alcohol dehydrogenase promoter reaching the concentration
of 25 mg/L (Ward et al., 1992b). Auxotrophic mutants of
Aspergillus strains bearing the defective prg4 gene were used
also for human Lf (hLf) production. Expression of this gene
gives the ability to produce an orotidine-50 -phosphate
(OMP) decarboxylase, the enzyme of uridine synthesis so
that the auxotrophic Aspergillus mutants cannot grow on
media lacking uridine (Conneely et al., 2001). Hence, the
presence of the OMP decarboxylase gene helps select
the transformed material on uridine-free medium. The
analysis of recombinant hLf puried from growth medium
of A. oryzae using CM Sephadex C50, SDS/PAGE
silver staining and Lf puried from human milk illustrated
that both proteins had similar N-glycosylation patterns
(Conneely et al., 2001).
Recombinant Lf expressed in A. niger var. awamori,
talactoferrin (Agennix, Inc.), is structurally and functionally
equivalent to native hLf and differs only in the nature of
its N-glycosylation (Hayes et al., 2005). Mutagenesis of
A. awamori strains that produce 250 mg/L hLf resulted in the
secretion of 2 g/L of hLf (Ward et al., 1995). As for
glycosylation, A. awamori-derived hLf contained high
mannose type of N-linked oligosaccharides in contrast to
complex carbohydrate structure of human milk Lf, which
nevertheless has not affected its functional activity (Ward
et al., 1995). Thus, lamentous fungi are unable to provide
the proper folding of non-fungal proteins (Jalving, 2005) and
is aggravated by the constant activity of proteases during the
secretion process, strikingly decreasing the protein yield
(van den Hombergh et al., 1997). As a result, complications
on both transcriptional (codon misusage, translocations,
and reduced mRNA stability) and translational (protein
folding, sorting, and protease susceptibility) levels occur
(Maras et al., 1999). Therefore, the development of the
alternative expression systems for pharmaceuticals and
nutrients production remains a relevant topic.
The most suitable candidates for the large-scale mammalian protein synthesis are higher plants and uni/multicellular algae, since they can provide N-glycosylation and other
eukaryotic post-translational modications required for a
full-edged protein activity, as well as the protection of
polypeptides from the proteolytic degradation (Franklin and
Mayeld, 2005; Breyer et al., 2009). Pioneering work
dedicated to hLf gene introduction into plant system was
done on tobacco cultivars (Mitra and Zhang, 1994). Among
the advantages of tobacco as a protein expression system is
the production of a bulk of green biomass, relatively short
992

vegetation period, and environmental safety, since tobacco


is neither a food nor a forage crop (Stefanova et al., 2008).
The cells of suspension tobacco culture line Nt-1 were
transformed with pAM1401 plasmid carrying hLf gene
under the control of 35S promoter, and callus expressing hLf
gene was obtained (Mitra and Zhang, 1994). Actual cytosolic
Lf concentration in individual transformed cells varied from
0.6% to 2.5% of total protein. This plant-derived hLf has the
same N-terminus as rhLf, binds equal amount of iron and
inhibits the human pathogens growth as native milk Lf does.
Tobacco-derived Lf is more efcient against such bacterial
phytopathogens as Xanthomonas campestris pv. phaseoli,
Pseudomonas syringae pv. phaseolicola, P. syringae pv.
syringae and Clavibacter accumfaciens pv. accumfaciens
in comparison to commercial Lf that might be explained by
the increase of its toxicity after the expression in plant system
(Mitra and Zhang, 1994). Considerably high (500 mg)
concentration of commercially available Lf had only 10%
of the tobacco-derived Lf antibacterial activity (Mitra and
Zhang, 1994).
These data on both hLf expression efciency and protein
activity corroborate the results of Choi et al. (2003), which
provide evidence for the expression level of part-length
(48 kDa) hLf ranging from 0.7% to 2.7% of total soluble
protein in obtained transgenic tobacco cell suspension lines.
Extracts of pooled calli prepared by Mitra and Zhang (1994)
expressed 1.8% Lf protein on average.
Later, transgenic tobacco plants expressing full-length hLf
gene were obtained (Salmon et al., 1998; Zhang et al., 1998b;
Liu et al., 1999, 2004). Maximum expression level of the
recombinant hLf in tobacco plants was in the range from
0.1% to 0.3% of total leaf protein (Salmon et al., 1998).
Furthermore, the N-terminal sequencing of the isolated
protein indicates that Lf molecules were correctly processed
(Salmon et al., 1998), data that agrees with Zhang et al.
(1998b) where hLf protein concentrations ranged from 0.1%
to 0.8% of total soluble protein. The expression level of
hLf N-lobe conferring Lf bactericidal properties in transgenic N. benthamiana plants transformed by agroinfection
amounted to 0.6% (~9 mg in 1.5 mg) of total soluble protein
(Li et al., 2004). Although several efcient hLf expression
systems for tobacco calli and suspension cells were
developed, this plant species is considered unsuitable for
the commercial production of plant-derived hLf because
of the presence of nicotine-related alkaloids (nicotine,
nornicotine, anabasine, and anatabine) and other healththreatening compounds (Stefanova et al., 2008). Another
disadvantage of this plant system is related to its harvesting,
transportation and storage, as the protein stability of
harvested material is low and it must be processed
immediately after gathering (Fischer et al., 2004).
A suitable candidate for the development of a new and
efcient system for rhLf production is alfalfa, characterized

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A.I. Yemets et al.

by a high biomass production at low cost, reduced


fertilization need, lack of toxic compounds, high vitamin,
mineral, and protein content (Stefanova et al., 2008, 2012).
In spite of hLf gene transfer conrmation into transgenic
alfalfa plants, expression of target sequence was detected
only in one clone. Quantitative analysis of this transgenic
clone revealed that full-length hLf content was only 0.0047%
of total soluble protein, which was considerably low even in
comparison with the results obtained by Salmon et al.
(1998). Interestingly, another transgenic clone in which
mhLf RNA were not detected expressed the recombinant
protein, but at a lower level (0.0035% of total soluble
protein). In the other tested clones recombinant protein was
not detected (Stefanova et al., 2012). However, despite the
certain advantage of the mentioned system, questions about
protein stability in harvested material remain.
The next step of plant-based Lf biofactories development
was its introduction into such edible food plants as potato
(Chong and Langridge, 2000). For this purpose, fusion gene
hlf-sekdel has been inserted into plant expression vector to
enhance the expression of target protein (Chong and
Langridge, 2000). Despite the use of two strong promoters,
constitutive 35S CaMV and auxin-inducible mas P2, the
amounts of full-length Lf in transgenic potato plants varied
from 0.01% to 0.1% of total soluble protein. However, Lf
expression in transgenic potato plants under mas P2
promoter was ~10-fold higher than the amount of Lf
generated under the enhanced 35 S CaMV promoter (Chong
and Langridge, 2000). Potato-produced hLf was also active
against E. coli (Migula) (ATCC 35218), E. coli (DH5a) and
Salmonella paratyphi. Though the results are very promising, classically potato must be cooked before eating because
of solanin accumulation, and it is unclear if the Lf protein
retains its biological activity after boiling (Lnnerdal, 2002;
Fischer et al., 2004; Stefanova et al., 2008).
Production of foreign proteins in plant cell cultures may
be more beneciary than in whole plants, because of time
constraints, better control and reproducible conditions in
bioreactors as compared to eld-grown transformed plants
(Min et al., 2006). Thus hLf has also been expressed in sweet
potato suspension culture cells under the control of 35S
CaMV promoter (~0.32% of total extracted protein) (Min
et al., 2006). Expression of hLf apparently did not inhibit cell
growth (Min et al., 2006). High level hLf production (up to
3% of total soluble protein) under an oxidative stressinducible peroxidase (SWPA2) promoter was reached in
ginseng suspension culture cells (Kwon et al., 2003).
Molecular analysis of transgenic P. ginseng suspension cells
showed the expression of full-length 80 and 40 kDa Lf. As
three calli lines produced only 80 kDa hLf, it was suggested
that part-length 40 kDa Lf is the result of the overexpressed
full-length Lf degradation during the extraction or termination of the premature protein synthesis (Kwon et al., 2003).

Plant-based biopharming of recombinant human Lf

Similar results on both Lf expression level and both full- and


part-length proteins were obtained with Siberian ginseng
culture cells under the same promoter (Jo et al., 2006).
However, contrary to previous results highlighting Siberian
ginseng transformation, an endoplasmic reticulum-targeting signal peptide was fused to hLf cDNA (Jo et al., 2006).
The growth patterns of non-transformed and transgenic cell
lines were almost similar, although an 8-day delay occurred
after the non-transformed cell line subcultivation. Accumulation of hLf in ginseng transgenic line increased from the
16th day after the subcultivation, reaching a maximum level
on the 28th day and yielding 3.6% of total soluble protein.
Further cultivation resulted in decreased hLf content (Jo
et al., 2006). Puried rhLf (500 mg) from Siberian ginseng
culture reduces the number of S. aureus (KCTC1916) and E.
coli DH5a colonies more intensely than the commercial hLf
does (Jo et al., 2006).
Other promising systems for large-scale production and
accumulation of target recombinant proteins, including Lf,
are cereal grains. According to Huang et al. (2010), the seed
composition comprises between 0.1% and 20% of the total
solid weight of human food. Moreover, mature cereal grains
provide a suitable environment for the storage of the
recombinant proteins for 510 years (Ritala et al., 2008).
Cereal seeds are also free of toxic compounds in contrast to
tobacco leaves and potato tubers, which make them
appropriate for the application as food additives (Stefanova
et al., 2008).
Rice as a model monocots species is an attractive system
for the expression and accumulation of heterologous
proteins. In many countries, rice grains are the rst solid
baby food due to rice hypoallergenicity and commercial
availability (Nandi et al., 2002). Indeed, the proper choice of
the transcriptional regulatory region and signal sequence for
the recombinant protein to the protein storage body in the
plasmid construction could signicantly enhance the
ultimate yield of target peptides. Additionally, the promoter
shows specically unregulated activity during seed maturation (Huang et al., 2010) and codon optimization (Nandi
et al., 2002; Suzuki et al., 2003; Rachmawati et al., 2005;
Huang et al., 2010; Lee et al., 2010; Lin et al., 2010), which are
extra benets of this rice-based expression system. Codon
optimization by the replacement of either adenine or
thymine at the third position to cytosine or guanine resulted
in the increase of the recombinant hLf production level from
0.5 to 5.0 g/kg in the dehusked rice grains. Native Lf has
a26-linked neuraminic acid, b14-linked galactose and
a16-linked fucose glycans typical for mammals (Spik
et al., 1982), while rhLf from koshihikari rice cultivar has
a13-linked fucose and b12-linked xylose typical plant
glycans (Fujiyama et al., 2004).
Apart from the differences in glycosylation, biochemical
and physical studies have shown that rhLf from rice and hLf

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A.I. Yemets et al.

are very similar (Nandi et al., 2002; Conesa et al., 2010) and
rice-derived rhLf has the same N-terminus as hLf portion
(Nandi et al., 2002). Thus, all molecular tools described
above enhanced the rhLf expression level to 4.55.5 g/kg of
the selected O. sativa homozygous line (LF164) over nine
generations (Nandi et al., 2005). As a result, quantitative
analysis of this transgenic line indicated 0.5% rhLf in brown
rice our amounting to >25% of the total soluble protein.
Comparable data were reported by Lin et al. (2010), where
the concentration of hLf expressed in seeds under the
glutelin (Gtl) promoter reached 0.45% of the total dry weight
of the dehusked rice seeds. The N-terminal sequence of this
rice-derived hLf was identical to that of native hLf mature
protein (Lin et al., 2010) as in a previous study (Nandi
et al., 2002). A similar approach with codon optimization
and Gt1 promoter resulted in 93130 mg of total rhLf in the
transformed rice seeds, which was patented by Huang et al.
(2010). The comparably high rhLf expression level achieved
in rice grains provides a strong base for the development of
its low-cost downstream processing (Nandi et al., 2005).
For the investigation of other rice expression systems,
a range of different regulatory sequences were tested
(Rachmawati et al., 2005). Thus, the inuence of the
constitutive maize ubiquitin-1 promoter, sequence encoding
native hLf or rice glutelin signal peptides was examined on
the hLf expression in Javanica rice. The expression level of
hLf in the vegetative tissue of the transgenic plant was <0.8%
of total soluble protein, but it was signicantly higher in
seeds. The highest level of rhLf expression with rice glutelin
signal peptide reached 2.0 mg/g in dehusked seeds, while
recombinant hLf expression level with hLf signal peptide was
only 1.6 mg/g. Therefore, the use of the correct signal peptide
would be important to locate the target protein to the
appropriate protein body of endosperm transgenic rice seeds
that protects Lf from the protease digestion. The presence of
recombinant hLf in all parts of transgenic plants conrmed
the constitutivity of maize ubiquitin-1 promoter. Low
recombinant hLf expression level in vegetative part of rice
plants may be explained by the enhanced protein susceptibility to protease degradation. The level of the recombinant
hLf production in transgenic seeds reached 15% of the total
soluble protein compared to tissue-specic Gtl promoter use
(20%) (Nandi et al., 2002; Lin et al., 2010). Nevertheless, Nterminal amino acid of hLf was identical to that of milk hLf
(Rachmawati et al., 2005). Therefore, such an approach gives
an opportunity to use Lf expression not only for the
accumulation and further oral administration as food
additives or pharmaceuticals, but also for plant protection
against numerous pathogens (Nandi et al., 2002).
Another promoter was chosen to produce the porcine
full-length recombinant Lf (prLf) in Japonica rice cultivar
TNG67 (Lee et al., 2010). The expression level of porcine
rLf in rice bran under the rice actin promoter was estimated
994

as ~1% of the total extractable protein. This protein


expression level was equivalent to ~0.1% of rice bran weight
that was lower than the protein expression level under the
endosperm-specic Gtl promoter (Nandi et al., 2002; Lin
et al., 2010). As in case of maize ubiquitin-1 promoter usage
(Rachmawati et al., 2005), prLf expression was also detected
in vegetative parts of transgenic plants. Although the
glycosylation pattern of rhLf differed from that of native
human Lf, the rice bran-derived porcine Lf as a food
supplement affects growth and immune characteristics of
early weaned piglets (Lee et al., 2010).
Rice grains as bioreactors are supposed comparatively to
be an even more efcient plant expression system for Lf
production. Despite its numerous benets, rice has not been
cultivated traditionally in many countries. Alternative
systems for the expression, accumulation and storage of
rhLf target protein are barley and wheat seeds. Barley is
grown over a broader range of environmental conditions
than any other cereals and considered to be an important
crop in the northern regions of North America and Europe
(Ritala et al., 2008; Mrzov et al., 2014). Barley seeds
store 15% of the protein (by weight), while rice seeds only
store 78%. Therefore, the transformation of barley by the
hLf gene was successfully performed (Kamenarova
et al., 2007; Tanasienko et al., 2011). Since the amount of
rhLf protein accumulation reached only 3 ng/mg of total
soluble protein (Kamenarova et al., 2007), the range of other
commercial barley cultivars were tested (Tanasienko
et al., 2009; Yemets et al., 2012) and the novel selection
system for the efcient genetic transformation was developed (Tanasienko et al., 2011; Yemets et al., 2012). Using
perspective European barley cultivar Oksamytoviy and
triuralin as a promising selective agent, transgenic barley
lines expressing hLf gene were obtained (Tanasienko
et al., 2011; Yemets et al., 2012).
Transgenic wheat expressing Lf had 80 kDa bands
corresponding to the molecular weight of bLf (Han
et al., 2012), reaching a protein yield of 2167 ng/mg of
leaf tissue. Lf yield has not reached signicantly higher
values than those obtained for rice (Nandi et al., 2002;
Rachmawati et al., 2005), which could be explained by the
use of different promoters. Interestingly, the Lf mRNA levels
in these lines were almost the same, while the protein
levels were threefold higher. To conrm the functional
activity of total protein extracts from the transgenic wheat
leaves, an agar-gel diffusion inhibition assay was determined, showing severe reduction of fungal (Fusarium
graminearum Schwabe) growth in the presence of Lf protein
extracts from the transgenic wheat plants.
All existing non-animal Lf expression systems (Table 1)
need to be improved from the standpoint of their expression
efciency, purication procedure of target protein as well as
the sophisticated cultivation, harvesting and biological

Cell Biol Int 38 (2014) 9891002 2014 International Federation for Cell Biology

Cell Biol Int 38 (2014) 9891002 2014 International Federation for Cell Biology

O. sativa L.
O. sativa L.
O. sativa L.
Ginseng (Panax ginseng C.A.
Meyer)
Pear (Pyrus sp.)

O. sativa L.

Tomato (Lycopersicon
esculentum Mill.)
Rice (Oryza sativa L.)

N. tabacum L. cv. Xanthi


Tobacco (N. benthamiana
Domin.)
Potato (Solanum tuberosum L.)
hLf, 0.010.1% of total soluble
protein
hLf, data not shown

Plant

bLf, data not shown

Plant
Plant
Plant
Cell suspension culture
Plant

Cell culture

hLf, 0.55.0 g/kg of dehusked rice


grains
rhLf, 24% of the total soluble
protein
hLf, 2.0 mg/g of dehusked seeds
pLf, 0.1% of rice bran weight
hLf, 0.45% of total dry weight
hLf, 3% of total soluble protein

Plant

Plant

Plant
Plant

Plant

N. tabacum L.

hLf, 0.62.5% of total protein


hLf, 0.72.7% of total soluble
protein
hLf, 0.10.3% of total soluble
protein
hLf , 0.10.8% of total soluble
protein
bLf, data not shown
hLfN, 0.6% of total soluble protein

Suspension culture cells


Cell suspension culture
Plant

hLf, 2 g/L

hLf, 25 mg/L

hLf, 25 mg/L

pLfN, data not shown


LfcinB, 90 mg/L

rhLf, 99.8 mg/L

rhLf, 115 mg/L

hLf, 1.52 mg/L

Lactoferrin origin, expression level

Mycelia

Mycelia

N. tabacum L.

A. awamori
Higher plants
Tobacco (Nicotiana tabacum L.)
N. tabacum L.

A. nidulans

Mycelia

Cell suspension culture

Pichia methanolica

Filamentous fungi
Aspergillus oryzae

Cell suspension culture

Cell suspension culture

Type of culture

Pichia pastoris

Yeast
Saccharomyces cerevisiae

Species

Table 1 Comparative analysis of different lactoferrin expression systems.

Full-length protein

Full-length protein

Details

Full-length protein

Full-length protein

Full-length protein

Erwinia amylovora resistance

Pharmaceuticals
Food additives
Development of selection system
Expression systems development

Food additives

Pharmaceuticals

Bacterial wilt resistance

Food additives

Full-length protein
Full-length protein
Full-length protein
Full-length, part-length (40 kDa)
protein
Full-length, part-length (60 kDa)

Full-length protein

Full-length protein

Full-length

Full-length protein

Resistance to Ralstonia
Full-length, part-length (48 kDa)
solanacearum
protein
Resistance to Rhizoctonia solani Full-length protein
Expression systems development Full-length hLfN protein

Expression systems development Full-length protein

Expression systems development Full-length protein


Expression systems development Part-length (48 kDa) protein

Pharmaceuticals

Pharmaceuticals

Pharmaceuticals

Expression systems development Full-length protein


Full-length protein

Pharmaceuticals

Pharmaceuticals

Purpose of use

continued

Malnoy et al. (2003)

Rachmawati et al. (2005)


Lee et al. (2010)
Lin et al. (2010)
Kwon et al. (2003)

Suzuki et al. (2003)

Nandi et al. (2002)

Chong and Langridge


(2000)
Lee et al. (2002)

Nguyen et al. (2011)


Li et al. (2004)

Zhang et al. (1998b)

Salmon et al. (1998)

Mitra and Zhang (1994)


Choi et al. (2003)

Ward et al. (1992a),


Conneely et al. (2001)
Ward et al. (1992b),
Conneely et al. (2001)
Ward et al. (1995)

Liang and Richardson


(1993)
Kruzel and Zimecki (2002),
Jiang et al. (2008)
Choi et al. (2008), Jo et al.
(2011)
Shan et al. (2007)
Wang et al. (2007)

References

A.I. Yemets et al.


Plant-based biopharming of recombinant human Lf

995

996

Han et al. (2012)

Stefanova et al. (2012)

Wheat (Triticum aestivum L.)

Plant

bLf, 2167 ng/mg of leaf tissue

Full-length protein
Pseudomonas syringae pv.
syringae and Clavibacter
michiganensis resistance
Fusarium graminearum resistance Full-length protein

Expression systems development mRNA


Fungal infection resistance
Full-length protein

H. vulgare L.
Plant
Arabidopsis (Arabidopsis thaliana Plant
(L.) Heynh.)
Alfalfa (Medicago sativa L.)
Plant

hLf, 0.0047% of total soluble


protein

Tanasienko et al. (2011)


Nguyen et al. (2011)

Kamenarova et al. (2007)


Expression systems development Full-length protein

hLf, 3 ng/mg of total soluble


protein
hLf
bLf, data not shown

Jo et al. (2006)
Expression systems development Full-length (80 kDa), part-length
(35 kDa) protein
hLf, 3.6% of total soluble protein

Min et al. (2006)


protein
hLf, 0.32% of total soluble protein Expression systems development Full-length protein

Sweet potato (Ipomoea batatas Cell suspension culture


(L.) Lam.)
Siberian ginseng (Acanthopanax Cell suspension culture
senticosus (Rupr. & Maxim)
Harms.)
Barley (Hordeum vulgare L.)
Plant

Species

Table 1. (Continued)

Type of culture

Lactoferrin origin, expression level

Purpose of use

Details

References

Plant-based biopharming of recombinant human Lf

A.I. Yemets et al.

activity. Only two bioreactors O. sativa and A. niger are


already in commercial use; hence, the search for alternative
hosts for rhLf expression remains attractive for
biotechnologists.
Lactoferrin expression as a tool for the
enhancement of non-specic plant pathogen
resistance
In contrast to mammals, there is no mobile defensive cells
and adaptive somatic immune system in plants (Jo
et al., 2006; Jones and Dangl, 2006). Instead, they rely on
the innate immunity of single cell and on systemic signals
emanating from infection sites (Jo et al., 2006; Jones and
Dangl, 2006).
Due to multiple protective Lf properties, its genes are
desirable candidates for the introduction into the genomes
of economically important higher plant species for the
enhancement of their immune response. Several attempts to
strengthen plant pathogen resistance have been done by the
transfer of hLf gene into tobacco (Zhang et al., 1998b;
Nguyen et al., 2011), tomato (Lee et al., 2002), pear (Malnoy
et al., 2003), rice (Takes et al., 2005) and wheat (Han
et al., 2012). As a result, primary lines of the transformed
tobacco plants (T0) with different resistance to untimely wilt
caused by the bacteria Ralstonia solanacearum were
obtained, and the plants of XNC/hlf-T1-2 and XNC/hlfT1-8 transgenic lines showing the highest resistance could
produce normal seeds similar to wild-type plants (Zhang
et al., 1998b). Quantication of total Lf conrmed a positive
correlation between the enhanced plant pathogen resistance,
Lf expression and accumulation ranging from 0.1% to 0.8%
of total soluble protein (Zhang et al., 1998b). A lowmolecular weight together with the full-length (80 kDa) Lf
was also found in the highly resistant transgenic tobacco cells
expressing hLf (Mitra and Zhang, 1994). A certain amount
of Lf could be truncated or degraded during the transformation process (Zhang et al., 1998b).
Besides transgenic tobacco, resistance to R. solanacearum
was achieved in susceptible tomato L. esculentum cultivar
F7926-96 expressing the full-length hLf gene under the
control of 35S promoter (Lee et al., 2002). First transgenic
(T1) generation of Lf-expressing tomato plants possessed
bacterial wilt resistance on the early stages of infection (Lee
et al., 2002). However, the level of resistance strongly varied
among the individual tomato transformed plants that also
may occur due to Lf differential expression (Lee et al. 2002)
as compared to earlier results (Zhang et al., 1998b). The nontransgenic plants inoculated with R. solanacearum suspension (1  107108 CFU/mL) totally wilted on 1011th day
after the inoculation, while the wilting of LF-T1-29 and
LF-T1-34 Lf-expressing lines was signicantly delayed to 23
24th day. LF-T1-34 transgenic plants wilted 16 days later
Cell Biol Int 38 (2014) 9891002 2014 International Federation for Cell Biology

A.I. Yemets et al.

compared to control, whereas wilting of LF-T1-11 and LFT1-28 Lf-expressing tomato lines was delayed just for 4 days.
Nevertheless, 4455% of T2 generation maintained wilting
resistance to the fruit ripening stage after the bacterial
inoculation. Therefore, regardless of Lf resistance instability,
transformation of the susceptible tomato cultivars by hLf
gene gave substantial benets to transgenic plants in
comparison to non-transformed ones. Several edible and
commercial plant species were transformed also by bovine Lf
gene (bLf). For instance, the transformation of pear plants by
bLf conferred the resistance to re blight caused by bacteria
Erwinia amylovora (Malnoy et al., 2003). In this investigation, nine transgenic clones were obtained, but only six of
them underwent the detailed analysis of transgene expression and disease resistance as the remaining clones expressed
bLf. However, in vitro cultivated plants had higher Lf
transcript levels than acclimatized plants. Despite this, all
clones produced the expected Lf protein in vivo. In all
transgenic lines the reduction of infection symptoms of
shoots in vitro was 17%, rooted plants 30%, and grafted
plants 60%. Furthermore, increased in vitro resistance of the
transformed pear plants to other diseases Agrobacterium
tumefaciens-induced crown galls and Pseudomonas syringae-caused bacterial blast was achieved.
Lf-expressing rice plants were examined for disease
resistance in vitro and in vivo against three pathogens:
Rice dwarf virus, bacterium Burkholderia (Pseudomonas)
plantarii, and fungus Pyricularia oryzae (Takase et al., 2005).
Two transgenic O. sativa lines with constitutive expression
of hLf and hLfN (the N-lobe of hLf) were tested. This Nterminal protein domain could be released by pepsin
digestion of Lf as an antibacterial peptide called lactoferricin
(Bellamy et al., 1992), which proved to be more potent a
bactericidal agent than Lf itself (Tomita et al., 1991). Both
hLf- and hLfN-transformed rice plants produced up to
0.1 mg of hLf/hLfN protein per 1 g of leaves. Because of the
signicant difference in molecular weights of plant-derived
full-length Lf and milk hLf, the comparative analysis of these
proteins was performed. It was shown that the rice-derived
hLf contains plant-type oligosaccharide chains, which may
partially account for the difference in milk hLf and rice hLf
molecular weights. The antibacterial resistance assay showed
the resistance of hLf- and hLfN-transformed rice plants to B.
plantarii, which causes blight in the seedlings. Some hLftransformed rice plants infected with Rice dwarf virus
showed a delay in symptoms manifestation; however, this
observation could be explained not with the enhancement of
hLf-transformants resistance, but with the delay of their
growth.
The retardation of Lf-expressing tobacco and pear growth
as compared to non-transformed plants was shown
previously (Zhang et al., 1998b; Malnoy et al., 2003). The
reduction of the total pear tree height was ~30% (Malnoy

Plant-based biopharming of recombinant human Lf

et al., 2003), the hLf-expressing tobacco plants ~10% (Zhang


et al., 1998b) and the hLf-expressing rice plants 1020%
(Takes et al., 2005). In turn, no direct or indirect effects of Lf
or its fragments were found in vitro and in vivo on O. sativa
resistance to the rice blast disease caused by P. oryzae (Takes
et al., 2005). As for the fungal infection, the transgenic
N. tabacum var Xanthi and A. thaliana plants expressing bLf
under 35S promoter showed resistance against fungal
pathogen Rhizoctonia solani, the causal agent of economically unfavorable damping-off plant diseases (Nguyen
et al., 2011). The transgenic plants are characterized by
the presence of 77 kDa Lf protein corresponding to the
molecular weight of full-length protein. Tobacco leaf
bioassay also showed plant resistance to the fungal pathogen.
As for Arabidopsis, the T2 transgenic generation seeds were
germinated in pots containing R. solani cornmeal mycelial
inoculums. Most seedlings in the control pots died out soon
after the germination, while the majority of the transgenic
seedlings grew normally, which indicates the successful gain
of resistance against R. solani-induced damping-off. Finally,
R. solani resistance was conrmed in all bLf-expressing
Arabidopsis and tobacco lines (Nguyen et al., 2011).
A bLf has also been introduced into a wheat cultivar,
Bobwhite, susceptible to fusarium head blight (FHB) caused
by the fungus Fusarium graminearum (Han et al., 2012). The
level of full-length target protein in T8 progeny of transgenic
lines varied from 21 to 67 ng/mg of leaf tissue. It was found
that Lf inhibits the growth of fungi in transgenic plants
both in vitro and in vivo. Variation in the resistance levels
among the independent transformation events correlated
with the actual amount of Lf in the transgenic lines, which
corroborates earlier results (Malnoy et al., 2003). The glumes
were infected during the manual inoculation of wheat
inorescence with a spore suspension, which could be
explained by the difference in concentrations of bLf in the
glumes and leaves (0.11% and 0.52% of the total soluble
protein, respectively). Nevertheless, all transgenic lines
showed a signicant level of the resistance as compared
to the untransformed Bobwhite, susceptible (Wheaton)
and resistant (ND 2710) to FHB wheat lines (Han et al.,
2012).
Alfalfa plants expressing hLf displayed mild symptoms of
Pseudomonas syringae pv. syringae infection in comparison
with non-transformed plants (Stefanova et al., 2012).
Moreover, the inoculation of fresh alfalfa branches with
Clavibacter michiganensis caused different level of symptoms reduction (Stefanova et al., 2012) that could be
explained by different Lf content.
Eventually, the transformation of some higher plant
species by a set of Lf genes (bLf, hLf, and hLfN) confers
broad-spectrum resistance against viral, bacterial, and fungal
plant diseases (Zhang et al., 1998b; Liu et al., 1999; Lee
et al., 2002; Takes et al., 2005) and is a promising tool for the

Cell Biol Int 38 (2014) 9891002 2014 International Federation for Cell Biology

997

Plant-based biopharming of recombinant human Lf

A.I. Yemets et al.

enhancement of plant biotic stress resistance crucial for food


production safety.
Important aspects of biosafety in lactoferrinproducing plants
The production of heterologous proteins in plant-based
expression systems also known as molecular farming
involves the use of genetically modied (GM) plants that
are obliged to undergo health and environmental risks
assessment (Ma et al., 2003; Breyer et al., 2009). Biological
safety of GM plants can be achieved by the application of
new vector constructs carrying health- and environmentally-friendly selective marker genes based exclusively on
genetic information already present in host plant, for
instance, mutant a-tubulin gene instead of widespread
antibiotic resistance genes (Blume et al., 2008; Yemets
et al., 2008). Recently, hLf-expressing transgenic barley was
obtained via biolistic transformation using plasmid vector
carrying this mutant a-tubulin gene providing resistance to
dinitroaniline herbicides (Tanasienko et al., 2011).
The main environmental risks remain foreign gene ow
from pollen/seed dispersal, horizontal gene transfer, as well
as potentially toxic effects of recombinant proteins on
symbiotic microorganisms, pollinating insects and herbivores (Ma et al., 2003). Hence, eld growing of GM plants or
the usage of such plants outside the constraints of physical
containment has to be authorized by Animal and Plant
Health Inspection Service (APHIS) in the USA (Breyer
et al., 2009). As for Lf production, only rice-based expression
system (Ventria Bioscience, USA) has been authorized by
APHIS for the treatment of iron deciency and acute
diarrhea (Bethell and Huang, 2004). For the appropriate
authorization also Standard Operating Procedures (SOPs,
USA) with the detailed instructions and additional guidance
for eld tests exists (USDA, 2005 Environmental Assessment). According to this document, rhLf synthesized in
O. sativa ssp. japonica cv. Taipei 309 is allowed to be
cultivated, harvested, stored and transported only after the
physical separation from the conventional rice crops by
equipment not used for the production or storage of a
commercial food rice. Moreover, the rice-derived rhLf has
been marked as Generally Recognized as Safe (GRAS), that
means its intentional addition to food after subjecting to
premarket review and approval of qualied experts of Food
and Drug Administration (FDA) as having been shown
adequately to be safe under the conditions of its intended use
(Bethell, 2004). Since transgenic rice was used for large-scale
hLf production, one of the major environmental safety
concerns is the possibility of transgenic hLf-expressing rice
escaping into the environment (Lin et al., 2010). A new
strategy for controlling the spread of hLf-expressing rice was
proposed based on the use of RNA interference cassette that
998

suppresses the expression of rice enzyme able to detoxify the


herbicide bentazon tagged with gene of interest. Different
cassettes carrying the desirable traits (hLf expression,
enhancement of bentazon sensitivity and glyphosate resistance for the transformants selection) were located in a
single T-DNA fragment that allows separating of one or two
trait(s) during the transformation. To test this, both
homozygous and heterozygous transgenic plants were
treated by 1000 mg/L bentazon, half its recommended
dose for rice weed control. The sensitivity of all plants
to bentazon treatment suggested the inheritance of all
three traits in case of transgenic and nontransgenic rice
hybridization (Lin et al., 2010). Thus the approach described
could potentiate the separation of Lf-expressing GM
plants in the environment and, therefore, decrease of
environmental risks.
Conclusions and future prospects
As natural sources of pharmaceutical proteins and other
bioactive molecules are rather limited, there is a growing
interest to the development of novel bioreactors for the
production of pharmaceuticals. Plant-based expression
systems have a set of advantages such as sufcient
polypeptides expression, eukaryotic protein folding and
relatively simple process of target protein purication in
comparison with yeast and fungal systems, allowing the
development of highly efcient technologies for the largescale production of Lf. Among all engineered plant-systems
for Lf synthesis the most promising ones are based on
rice. Microalgae (Franklin and Mayeld, 2005), barley
(Kamenarova et al., 2007; Tanasienko et al., 2009, 2011;
Yemets et al., 2012) and alfalfa (Stefanova et al., 2013) could
also be used as alternative platforms for the accumulation of
high levels of the recombinant proteins.
Acknowledgement
We appreciate suggestions from Andrej. S. Mosyakin
(Institute of Botany, National Academy of Sciences of
Ukraine, Kiev) about the improvement of manuscript
language.

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Received 3 October 2013; accepted 31 March 2014.
Final version published online 19 May 2014.

Cell Biol Int 38 (2014) 9891002 2014 International Federation for Cell Biology