Cell Biology International ISSN 1065-6995doi: 10.1002/cbin.10304

REVIEW

Plant-based biopharming of recombinant human lactoferrinAlla 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 thepromising human milk proteins, occupies the expanding biotechnological food market niche due to its important versatileproperties. Lf shows antiviral, antimicrobial, antiprotozoal and antioxidant activities, modulates cell growth rate, bindsglycosaminoglycans and lipopolysaccharides, and also inputs into the innate/specific immune responses. Development ofhighly efficient human recombinant Lf expression systems employing yeasts, filamentous fungi and undoubtedly higherplants as bioreactors for the large-scale Lf production is a biotechnological challenge. This review highlights the advantagesand disadvantages of the existing non-animal Lf expression systems from the standpoint of protein yield and its biologicalactivity. Special emphasis is put on the benefits of monocot plant system for Lf expression and the biosafety aspects of thetransgenic Lf-expressing plants.

Keywords: human lactoferrin; recombinant expression systems; plant-based biofarming; plant-pathogen resistance;biosafety

Introduction

Recently numerous recombinant proteins have been usedintensely in pharmacy, industry and research and, therefore,have to meet a range of sophisticated quality requirements,before they could be considered safe, in particular, anextra high-purity (Ma et al., 2003). According to GoodManufacturing Practice, all recombinant proteins must besufficiently pure and homogeneous with contaminantsremoved to acceptable levels (Fischer et al., 2012). It isimportant either to improve the protein production fromtheir native sources or to search for new ones together withthe development of the efficient protein expression systemsand the advance of protein extraction protocols.

Novel recombinant proteins, also referred to as “high-molecular drugs”, could be the targeted agents for thetreatment of such common health problems of industrialcountries as oncological, cardiovascular and infectiousdiseases—all critical to an expanding and aging humanpopulation (Elbehri, 2005). The existing pharmaceutical

industry is based on the chemical synthesis and/orproduction of the organic molecules by transgenic micro-organisms, mammalian cell cultures, or animals (Schwartz,2001;Ma et al., 2003). However, the eukaryotic folding of therecombinant proteins and their proper posttranslationalmodifications (e.g., glycosylation and phosphorylation)are the basic prerequisites for the protein biological activitythat cannot be ensured into the transgenic prokaryotes(Houdebaine, 2000; Ma et al., 2003). Mammalian cellcultures and transgenic animals also have such numerousdisadvantages as time-consuming protein expression,unprofitable purification procedure and, additionally, therisk of contamination with viral and oncogenic DNAs(van Berkel et al. 2002; Stefanova et al., 2008). The promisingtrend in biotechnology is the use of plants as “greenbioreactors” for recombinant protein production. At thesame time, the key advantages of plant expressionsystems are sufficient protein yield, eukaryotic proteinfolding along with comparatively short life cycles, easy seedstorage, absence of animal, and human viruses, and, last

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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 theconventional “first range proteins” for biopharming due toits numerous biological activities (Stefanova et al., 2008). Lf,a 80 kDa globular protein from the transferrins family, isproduced by mucosal gland cells of various mammalianspecies, and can be found in all secretory fluids (milk,colostrum, saliva, tears, etc.) (Adlerova et al., 2008; González-Chávez et al., 2009; Lönnerdal and Suzuki, 2013). Apart fromits main physiological functions, namely binding andtransport of iron ions, Lf exhibits antiviral, antimicrobial,antiprotozoal and antioxidant activities. It is also able tomodulate cell growth rate and to bind viral glycosaminogly-cans and bacterial lipopolysaccharides (Wakabayashi et al.,2006). Pepsin digestion of Lf generates its fragmentlactoferricin (Bellamy et al., 1992) that also reveals potentbactericidal activity against antibiotic-resistant strains ofStaphylococcus aureus and Escherichia coli from clinicalorigins (Flores-Villase~nor et al., 2010). Moreover, recombi-nant Lf has been tested for clinical use in treatment andprevention of such human and animal diseases as solidtumors (Hayes et al., 2005, 2010) and diarrhea (Humphreyet al., 2002). Its use as vaccine adjuvant modulates theadaptive immune response in humans (Hwang et al., 2011).

Lf is an iron-binding protein, and its functions can berelated to this property (Lönnerdal and Suzuki, 2013). Thus,one of the mechanisms of Lf antimicrobial properties isbased on its ability to sequester iron ions from the bacterialpathogens (García-Montoya et al., 2012). Lf can eliminatemicroorganisms via iron-independent pathway (Valenti andAntonini, 2005) by the direct interaction with the bacterialcell surface (Bortner et al., 1989; Farnaud and Evans, 2005;García-Montoya et al., 2012) and release lipopolysaccharide(LPS) from the cell wall of Gram-negative bacteria causing“poration” that allows exposure of the inner membraneproteoglycan layer to lysozyme activity (Ellison andGiehl, 1991; Lönnerdal and Suzuki, 2013).

Lf has antiviral activity against a broad range of RNA andDNA-containing human and animal viruses (García-Montoya et al., 2012; Lönnerdal and Suzuki, 2013) byinhibiting virus–host interaction, virus trafficking or directbinding of the viral particle by the blocking of glycosamino-glycan viral receptors, especially heparan sulfate (García-Montoya et al., 2012). It acts strongly against HIV in vitro(García-Montoya et al., 2012) by inhibiting viral replicationinside the host cell (Swart et al., 1996; Qiu et al., 1998;García-Montoya et al., 2012).Moreover, the interaction of Lfwith nucleolin surface blocks the attachment and entry ofHIV particles into HeLa P4 cells (Legrand et al., 2004).

Lf also possesses antifungal activity (Gifford et al., 2005)by its direct interplay with the pathogen and Fe3þ

sequestration (Zarember et al., 2007; González-Chávezet al., 2009). Thus, Lf eliminates Candida albicans andC. krusei by the alterating the permeability of their cellsurfaces (Wakabayashi et al., 1996; García-Montoya et al.,2012).

Many reports indicate the beneficial effects of bovine andhuman Lf in cancer treatment (Gibbons et al., 2011;Vogel, 2012), including chemically induced tumors inlaboratory rodents (Adlerova et al., 2008). Lf prevents cellcycle transitions from G1 to S (Damiens et al., 1999) and G0

to G1 phases (Xiao et al., 2004), and modulates cytokineproduction in malignant cells (García-Montoya et al., 2012).It can promote apoptosis and arrest tumor growth in vitro(Lönnerdal and Suzuki, 2013). Among the other factorsassociated with Lf’s anticancer effects are the down-regulation of phase I detoxifying enzyme and cytochromeP450 1A2 (Fujita et al., 2002), and the upregulation of phaseII detoxifying enzyme and glutathione-S-transferase, with aconsequent decrease in carcinogen activation (Tanakaet al., 2000).

Fluctuation in Lf content may be also used as biomarkerfor disease indication (Vogel, 2012), such as chronicperiodontitis (Glimvall et al., 2012), ulcerative colitis andCrohn’s disease (Vogel, 2012). Though Lf levels areincreased in the synovial fluid of the inflamed knee joints,suggesting neutrophil infiltration, the Lf levels in serumwere indistinguishable from healthy controls (Caccavoet 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 acidcontent after fat overload (Fernandez-Real et al., 2010;Lönnerdal and Suzuki, 2013), suggesting an importantrole of Lf in fat metabolism due to its antiadipogenic,antioxidative and anti-inflammatory activities (Lönnerdaland Suzuki, 2013).

Despite numerous beneficial effects of this multifunc-tional protein in the treatment of various infectious diseases,little is understood about its mechanisms of action.

Comparative characteristics of the existingeukaryotic non-animal systems of lactoferrinexpression

Recombinant Lf (rLf) has been expressed in a range oforganisms 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 andRichardson, 1993; Conneely et al., 2001); filamentous fungi:Aspergillus oryzae, A. nidulans (Conneely et al., 2001; Ward

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et al., 1992a) and A. awamori (Ward et al., 1995); higherplants: tobacco (Nicotiana tabacum L.) (Salmon et al., 1998)and Australian tobacco (N. bentamiana Domin.) (Liet al., 2004), potato (Solanum tuberosum L.) (Chong andLangridge, 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 ginsengC.A. Meyer) (Kwon et al., 2003), sweet potato (Ipomoeabatatans (L.) Lam.) (Min et al., 2006), eleuthero (Acantho-panax senticosus (Rupr. & Maxim) Harms) (Jo et al., 2006),Thale cress (Arabidopsis thaliana (L.) Heynh.) (Nguyenet al., 2011), barley (Hordeum vulgare L.) (Tanasienkoet 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.) (Liuet al., 2005); mammals: cow (Bos sp. Bojan.) (van Berkelet al., 2002) and goat (Capra sp. L.) (Han et al., 2007). In spiteof the large list of rLf expressing systems, only a few havebeen approved for the market and/or introduced in clinicalpractice. For instance, Aspergillus niger-produced Lf (tradename – talactoferrin) (Ward et al., 1995) is used for solidtumors treatment (Hayes et al., 2005, 2010) and Oryzasativa-derived Lf (trade name – lacromin) is an anti-apoptotic cell culture media supplement that increases cellgrowth rate (InVitria, Ventria Bioscience, USA, http://www.invitria.com). In this review, the advantages and disadvan-tages of yeast, fungal, and plant expression systems forrecombinant human Lf production (rhLf) are discussedtouching upon Lf biosafety aspects. The up-to-date approachconcerning the use of Lf plant expression systems for theenhancement of non-specific plant pathogen resistance isalso highlighted.

Such mammalian expression systems as cow (van Berkelet al., 2002) and goat (Han et al., 2007) have numerousdisadvantages, including potential risk of viral contamina-tion of target proteins, long life cycle, expensive purificationprocedures, as well as bioethical concerns (Stefanovaet al., 2008). Therefore, new Lf sources and/or biofactoriesenabling high expression levels of target protein for its large-scale production, easy extraction and purification proce-dures are required. For this reason, such widely usedproducers as yeast, filamentous fungi, and higher plants arebeing considered as the most efficient eukaryotic systems forLf expression.

Yeast have been used since ancient times as universalbioreactors suitable for pharmaceutical and nutrient proteinproduction because of their eukaryotic protein foldingsystem, simplicity of cultivation and common protocols forgene manipulations in the majority of unicellular organisms(Cereghino and Cregg, 1999). The range of the advantages,such as target gene expression under the strong regulatedalcohol oxidase I (AOX1) promoter, marker/host strain

combination and also the high cell culture density, makeP. pastoris an efficient bioreactor for recombinant human Lfproduction (Cereghino and Cregg, 1999; Kruzel andZimecki, 2002; Jo et al., 2011). In order to increase hLfexpression in P. pastoris, a codon-optimized hLf gene wasfused to 11 different signal sequences for the identification ofthe optimal one that facilitates the translocation of rhLf intothe secretory pathway and finally into the culture medium(Choi et al., 2008). From all tested S. cerevesiae signalsequences, alpha mating factor prepro has been selectedand modified to facilitate the processing of the proteinprior to mature rhLf secretion. The resulting sequence(ScaMFppKR) led not only to the facilitation of hLfsecretion, but it also minimized its intracellular accumula-tion. Two promoters –widely used in yeast inducible alcoholoxidase 1 (pAOX1) (Cereghino and Cregg, 1999, 2000) andconstitutive P. pastoris glyceraldehyde-3-phosphate dehy-drogenase (PpGAPDH) – were also tested for their ability toexpress rhLf. The combination of AOX1 promoter, codon-optimized hLf gene and ScaMFppKR signal sequence fromS. cerevesiae allowed 99.8mg/L of Lf to be reached (Choiet al., 2008). Moreover, the microbial surface displayapproach has been proposed to increase hLf level basedon its expression in P. pastoris, with the consequentimmobilization of the protein on the cell surface (Joet al., 2011). The hLf gene was fused to glycosylphospha-tidylinositol (GPI)-anchored protein of S. cerevisiae as ananchoring motif and expressed under the control of theAOX1 promoter. Analysis of this expression systemconfirmed the localization of rhLf in P. pastoris membraneas an integral protein closely associated with other cellularproteins (Jo et al., 2011). Lf produced in P. pastoris does notundergo core-fucosylation of N-linked glycans typical ofhuman neutrophilic leukocytes, whereas human milk-derived Lf displays fucose residues on N-acetylglucosamine(Choi et al., 2008).

In spite of ancient biotechnological traditions and evidentprogress in genetic and molecular biology, the use of S.cerevisiae as Lf expression system has some limitationsconcerning its lower secretory capacity compared with otheryeasts species (Cereghino and Cregg, 1999). Nevertheless,the optimization of the vector construction via invertasesignal sequence enhanced the hLf yield in S. cerevisiae to1.5–2mg/L (Liang and Richardson, 1993). However, thesignificant disadvantage of yeasts as heterologous proteinbioreactors is their inability to provide proper eukaryoticprotein post-translational modifications as amidation andprolylhydroxylation, and also some types of phosphoryla-tion and glycosylation (Cregg and Higgins, 1995).

In turn, the secretion potential of filamentous fungidistinguishes these organisms favorably from the otherperspective producers of bioactive proteins. Filamentousfungi, in contrast to prokaryotes, can provide proper

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eukaryotic post-translational modifications of polypeptidesas N-glycosylation and disulfide bonds formation (Maraset al., 1999). Lf expression in three filamentous fungi:A. oryzae under the control of a-amylase promoter and the30-flanking region of the A. niger glucoamylase gene, and inA. nidulans under the control of strong ethanol-induciblealcohol dehydrogenase promoter reaching the concentrationof 25mg/L (Ward et al., 1992b). Auxotrophic mutants ofAspergillus strains bearing the defective prg4 gene were usedalso for human Lf (hLf) production. Expression of this genegives the ability to produce an orotidine-50-phosphate(OMP) decarboxylase, the enzyme of uridine synthesis sothat the auxotrophic Aspergillus mutants cannot grow onmedia lacking uridine (Conneely et al., 2001). Hence, thepresence of the OMP decarboxylase gene helps selectthe transformed material on uridine-free medium. Theanalysis of recombinant hLf purified from growth mediumof A. oryzae using CM Sephadex C50, SDS/PAGEsilver staining and Lf purified from human milk illustratedthat 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 functionallyequivalent to native hLf and differs only in the nature ofits N-glycosylation (Hayes et al., 2005). Mutagenesis ofA. awamori strains that produce 250mg/L hLf resulted in thesecretion of 2 g/L of hLf (Ward et al., 1995). As forglycosylation, A. awamori-derived hLf contained highmannose type of N-linked oligosaccharides in contrast tocomplex carbohydrate structure of human milk Lf, whichnevertheless has not affected its functional activity (Wardet al., 1995). Thus, filamentous fungi are unable to providethe proper folding of non-fungal proteins (Jalving, 2005) andis aggravated by the constant activity of proteases during thesecretion process, strikingly decreasing the protein yield(van den Hombergh et al., 1997). As a result, complicationson both transcriptional (codon misusage, translocations,and reduced mRNA stability) and translational (proteinfolding, sorting, and protease susceptibility) levels occur(Maras et al., 1999). Therefore, the development of thealternative expression systems for pharmaceuticals andnutrients production remains a relevant topic.

The most suitable candidates for the large-scale mamma-lian protein synthesis are higher plants and uni/multicellu-lar algae, since they can provide N-glycosylation and othereukaryotic post-translational modifications required for afull-fledged protein activity, as well as the protection ofpolypeptides from the proteolytic degradation (Franklin andMayfield, 2005; Breyer et al., 2009). Pioneering workdedicated to hLf gene introduction into plant system wasdone on tobacco cultivars (Mitra and Zhang, 1994). Amongthe advantages of tobacco as a protein expression system isthe production of a bulk of green biomass, relatively short

vegetation period, and environmental safety, since tobaccois neither a food nor a forage crop (Stefanova et al., 2008).The cells of suspension tobacco culture line Nt-1 weretransformed with pAM1401 plasmid carrying hLf geneunder the control of 35S promoter, and callus expressing hLfgene was obtained (Mitra and Zhang, 1994). Actual cytosolicLf concentration in individual transformed cells varied from0.6% to 2.5% of total protein. This plant-derived hLf has thesame N-terminus as rhLf, binds equal amount of iron andinhibits the human pathogens growth as native milk Lf does.Tobacco-derived Lf is more efficient against such bacterialphytopathogens as Xanthomonas campestris pv. phaseoli,Pseudomonas syringae pv. phaseolicola, P. syringae pv.syringae and Clavibacter flaccumfaciens pv. flaccumfaciensin comparison to commercial Lf that might be explained bythe increase of its toxicity after the expression in plant system(Mitra and Zhang, 1994). Considerably high (500mg)concentration of commercially available Lf had only 10%of the tobacco-derived Lf antibacterial activity (Mitra andZhang, 1994).

These data on both hLf expression efficiency and proteinactivity corroborate the results of Choi et al. (2003), whichprovide evidence for the expression level of part-length(48 kDa) hLf ranging from 0.7% to 2.7% of total solubleprotein 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 hLfgene were obtained (Salmon et al., 1998; Zhang et al., 1998b;Liu et al., 1999, 2004). Maximum expression level of therecombinant hLf in tobacco plants was in the range from0.1% to 0.3% of total leaf protein (Salmon et al., 1998).Furthermore, the N-terminal sequencing of the isolatedprotein 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 ofhLf N-lobe conferring Lf bactericidal properties in trans-genic N. benthamiana plants transformed by agroinfectionamounted to 0.6% (~9mg in 1.5mg) of total soluble protein(Li et al., 2004). Although several efficient hLf expressionsystems for tobacco calli and suspension cells weredeveloped, this plant species is considered unsuitable forthe commercial production of plant-derived hLf becauseof the presence of nicotine-related alkaloids (nicotine,nornicotine, anabasine, and anatabine) and other health-threatening compounds (Stefanova et al., 2008). Anotherdisadvantage of this plant system is related to its harvesting,transportation and storage, as the protein stability ofharvested material is low and it must be processedimmediately after gathering (Fischer et al., 2004).

A suitable candidate for the development of a new andefficient system for rhLf production is alfalfa, characterized

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by a high biomass production at low cost, reducedfertilization need, lack of toxic compounds, high vitamin,mineral, and protein content (Stefanova et al., 2008, 2012).In spite of hLf gene transfer confirmation into transgenicalfalfa plants, expression of target sequence was detectedonly in one clone. Quantitative analysis of this transgenicclone revealed that full-length hLf content was only 0.0047%of total soluble protein, which was considerably low even incomparison with the results obtained by Salmon et al.(1998). Interestingly, another transgenic clone in whichmhLf RNA were not detected expressed the recombinantprotein, but at a lower level (0.0035% of total solubleprotein). In the other tested clones recombinant protein wasnot detected (Stefanova et al., 2012). However, despite thecertain advantage of the mentioned system, questions aboutprotein stability in harvested material remain.

The next step of plant-based Lf biofactories developmentwas its introduction into such edible food plants as potato(Chong and Langridge, 2000). For this purpose, fusion genehlf-sekdel has been inserted into plant expression vector toenhance the expression of target protein (Chong andLangridge, 2000). Despite the use of two strong promoters,constitutive 35S CaMV and auxin-inducible mas P2, theamounts of full-length Lf in transgenic potato plants variedfrom 0.01% to 0.1% of total soluble protein. However, Lfexpression in transgenic potato plants under mas P2promoter was ~10-fold higher than the amount of Lfgenerated under the enhanced 35 S CaMV promoter (Chongand Langridge, 2000). Potato-produced hLf was also activeagainst E. coli (Migula) (ATCC 35218), E. coli (DH5a) andSalmonella paratyphi. Though the results are very promis-ing, classically potato must be cooked before eating becauseof solanin accumulation, and it is unclear if the Lf proteinretains its biological activity after boiling (Lönnerdal, 2002;Fischer et al., 2004; Stefanova et al., 2008).

Production of foreign proteins in plant cell cultures maybe more beneficiary than in whole plants, because of timeconstraints, better control and reproducible conditions inbioreactors as compared to field-grown transformed plants(Min et al., 2006). Thus hLf has also been expressed in sweetpotato suspension culture cells under the control of 35SCaMV promoter (~0.32% of total extracted protein) (Minet al., 2006). Expression of hLf apparently did not inhibit cellgrowth (Min et al., 2006). High level hLf production (up to3% of total soluble protein) under an oxidative stress-inducible peroxidase (SWPA2) promoter was reached inginseng suspension culture cells (Kwon et al., 2003).Molecular analysis of transgenic P. ginseng suspension cellsshowed the expression of full-length 80 and 40 kDa Lf. Asthree calli lines produced only 80 kDa hLf, it was suggestedthat part-length 40 kDa Lf is the result of the overexpressedfull-length Lf degradation during the extraction or termina-tion of the premature protein synthesis (Kwon et al., 2003).

Similar results on both Lf expression level and both full- andpart-length proteins were obtained with Siberian ginsengculture cells under the same promoter (Jo et al., 2006).However, contrary to previous results highlighting Siberianginseng transformation, an endoplasmic reticulum-target-ing signal peptide was fused to hLf cDNA (Jo et al., 2006).The growth patterns of non-transformed and transgenic celllines were almost similar, although an 8-day delay occurredafter the non-transformed cell line subcultivation. Accumu-lation of hLf in ginseng transgenic line increased from the16th day after the subcultivation, reaching a maximum levelon the 28th day and yielding 3.6% of total soluble protein.Further cultivation resulted in decreased hLf content (Joet al., 2006). Purified rhLf (500mg) from Siberian ginsengculture reduces the number of S. aureus (KCTC1916) and E.coli DH5a colonies more intensely than the commercial hLfdoes (Jo et al., 2006).

Other promising systems for large-scale production andaccumulation of target recombinant proteins, including Lf,are cereal grains. According to Huang et al. (2010), the seedcomposition comprises between 0.1% and 20% of the totalsolid weight of human food. Moreover, mature cereal grainsprovide a suitable environment for the storage of therecombinant proteins for 5–10 years (Ritala et al., 2008).Cereal seeds are also free of toxic compounds in contrast totobacco leaves and potato tubers, which make themappropriate for the application as food additives (Stefanovaet al., 2008).

Rice as a model monocots species is an attractive systemfor the expression and accumulation of heterologousproteins. In many countries, rice grains are the first solidbaby food due to rice hypoallergenicity and commercialavailability (Nandi et al., 2002). Indeed, the proper choice ofthe transcriptional regulatory region and signal sequence forthe recombinant protein to the protein storage body in theplasmid construction could significantly enhance theultimate yield of target peptides. Additionally, the promotershows specifically unregulated activity during seed matura-tion (Huang et al., 2010) and codon optimization (Nandiet al., 2002; Suzuki et al., 2003; Rachmawati et al., 2005;Huang et al., 2010; Lee et al., 2010; Lin et al., 2010), which areextra benefits of this rice-based expression system. Codonoptimization by the replacement of either adenine orthymine at the third position to cytosine or guanine resultedin the increase of the recombinant hLf production level from0.5 to 5.0 g/kg in the dehusked rice grains. Native Lf hasa2–6-linked neuraminic acid, b1–4-linked galactose anda1–6-linked fucose glycans – typical for mammals (Spiket al., 1982), while rhLf from “koshihikari” rice cultivar hasa1–3-linked fucose and b1–2-linked xylose – typical plantglycans (Fujiyama et al., 2004).

Apart from the differences in glycosylation, biochemicaland physical studies have shown that rhLf from rice and hLf

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are very similar (Nandi et al., 2002; Conesa et al., 2010) andrice-derived rhLf has the same N-terminus as hLf portion(Nandi et al., 2002). Thus, all molecular tools describedabove enhanced the rhLf expression level to 4.5–5.5 g/kg ofthe selected O. sativa homozygous line (LF164) over ninegenerations (Nandi et al., 2005). As a result, quantitativeanalysis of this transgenic line indicated 0.5% rhLf in brownrice flour amounting to >25% of the total soluble protein.Comparable data were reported by Lin et al. (2010), wherethe concentration of hLf expressed in seeds under theglutelin (Gtl) promoter reached 0.45% of the total dry weightof the dehusked rice seeds. The N-terminal sequence of thisrice-derived hLf was identical to that of native hLf matureprotein (Lin et al., 2010) as in a previous study (Nandiet al., 2002). A similar approach with codon optimizationand Gt1 promoter resulted in 93–130mg of total rhLf in thetransformed rice seeds, which was patented by Huang et al.(2010). The comparably high rhLf expression level achievedin rice grains provides a strong base for the development ofits 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 influence of theconstitutive maize ubiquitin-1 promoter, sequence encodingnative hLf or rice glutelin signal peptides was examined onthe hLf expression in Javanica rice. The expression level ofhLf in the vegetative tissue of the transgenic plant was<0.8%of total soluble protein, but it was significantly higher inseeds. The highest level of rhLf expression with rice glutelinsignal peptide reached 2.0mg/g in dehusked seeds, whilerecombinant hLf expression level with hLf signal peptide wasonly 1.6mg/g. Therefore, the use of the correct signal peptidewould be important to locate the target protein to theappropriate protein body of endosperm transgenic rice seedsthat protects Lf from the protease digestion. The presence ofrecombinant hLf in all parts of transgenic plants confirmedthe constitutivity of maize ubiquitin-1 promoter. Lowrecombinant hLf expression level in vegetative part of riceplants may be explained by the enhanced protein suscepti-bility to protease degradation. The level of the recombinanthLf production in transgenic seeds reached 15% of the totalsoluble protein compared to tissue-specific Gtl promoter use(20%) (Nandi et al., 2002; Lin et al., 2010). Nevertheless, N-terminal amino acid of hLf was identical to that of milk hLf(Rachmawati et al., 2005). Therefore, such an approach givesan opportunity to use Lf expression not only for theaccumulation and further oral administration as foodadditives or pharmaceuticals, but also for plant protectionagainst numerous pathogens (Nandi et al., 2002).

Another promoter was chosen to produce the porcinefull-length recombinant Lf (prLf) in Japonica rice cultivarTNG67 (Lee et al., 2010). The expression level of porcinerLf in rice bran under the rice actin promoter was estimated

as ~1% of the total extractable protein. This proteinexpression level was equivalent to ~0.1% of rice bran weightthat was lower than the protein expression level under theendosperm-specific Gtl promoter (Nandi et al., 2002; Linet al., 2010). As in case of maize ubiquitin-1 promoter usage(Rachmawati et al., 2005), prLf expression was also detectedin vegetative parts of transgenic plants. Although theglycosylation pattern of rhLf differed from that of nativehuman Lf, the rice bran-derived porcine Lf as a foodsupplement affects growth and immune characteristics ofearly weaned piglets (Lee et al., 2010).

Rice grains as bioreactors are supposed comparatively tobe an even more efficient plant expression system for Lfproduction. Despite its numerous benefits, rice has not beencultivated traditionally in many countries. Alternativesystems for the expression, accumulation and storage ofrhLf target protein are barley and wheat seeds. Barley isgrown over a broader range of environmental conditionsthan any other cereals and considered to be an importantcrop in the northern regions of North America and Europe(Ritala et al., 2008; Mrízová et al., 2014). Barley seedsstore 15% of the protein (by weight), while rice seeds onlystore 7–8%. Therefore, the transformation of barley by thehLf gene was successfully performed (Kamenarovaet al., 2007; Tanasienko et al., 2011). Since the amount ofrhLf protein accumulation reached only 3 ng/mg of totalsoluble protein (Kamenarova et al., 2007), the range of othercommercial barley cultivars were tested (Tanasienkoet al., 2009; Yemets et al., 2012) and the novel selectionsystem for the efficient genetic transformation was devel-oped (Tanasienko et al., 2011; Yemets et al., 2012). Usingperspective European barley cultivar Oksamytoviy andtrifluralin as a promising selective agent, transgenic barleylines expressing hLf gene were obtained (Tanasienkoet al., 2011; Yemets et al., 2012).

Transgenic wheat expressing Lf had 80 kDa bandscorresponding to the molecular weight of bLf (Hanet al., 2012), reaching a protein yield of 21–67 ng/mg ofleaf tissue. Lf yield has not reached significantly highervalues than those obtained for rice (Nandi et al., 2002;Rachmawati et al., 2005), which could be explained by theuse of different promoters. Interestingly, the Lf mRNA levelsin these lines were almost the same, while the proteinlevels were threefold higher. To confirm the functionalactivity of total protein extracts from the transgenic wheatleaves, an agar-gel diffusion inhibition assay was deter-mined, showing severe reduction of fungal (Fusariumgraminearum Schwabe) growth in the presence of Lf proteinextracts from the transgenic wheat plants.

All existing non-animal Lf expression systems (Table 1)need to be improved from the standpoint of their expressionefficiency, purification procedure of target protein as well asthe sophisticated cultivation, harvesting and biological

Plant-based biopharming of recombinant human Lf A.I. Yemets et al.

994 Cell Biol Int 38 (2014) 989–1002 © 2014 International Federation for Cell Biology

Table

1Comparativean

alysisofdifferentlactoferrin

expressionsystem

s.

Species

Type

ofcultu

reLactoferrin

origin,expression

level

Purposeof

use

Details

References

Yeast

Saccha

romyces

cerevisiae

Cellsuspe

nsioncultu

rehLf,1.5–

2mg/L

Pharmaceu

ticals

Full-leng

thprotein

Lian

gan

dRichardson

(199

3)Pichia

pastoris

Cellsuspe

nsioncultu

rerhLf,11

5mg/L

Pharmaceu

ticals

Full-leng

thprotein

Kruzeland

Zimecki(200

2),

Jiang

etal.(200

8)rhLf,99

.8mg/L

Cho

ietal.(200

8),Joet

al.

(201

1)Pichia

metha

nolica

Cellsuspe

nsioncultu

repLfN,da

tano

tshow

nExpression

system

sde

velopm

ent

Full-leng

thprotein

Shan

etal.(200

7)LfcinB

,90

mg/L

Full-leng

thprotein

Wan

get

al.(200

7)Filamen

tous

fung

iAspergillusoryzae

Mycelia

hLf,25

mg/L

Pharmaceu

ticals

Full-leng

thprotein

Wardet

al.(199

2a),

Con

neelyet

al.(200

1)A.nidu

lans

Mycelia

hLf,25

mg/L

Pharmaceu

ticals

Full-leng

thprotein

Wardet

al.(199

2b),

Con

neelyet

al.(200

1)A.aw

amori

Mycelia

hLf,2g/L

Pharmaceu

ticals

Full-leng

thprotein

Wardet

al.(199

5)Highe

rplan

tsTo

bacco(Nicotiana

taba

cum

L.)

Suspen

sion

cultu

recells

hLf,0.6–

2.5%

oftotalp

rotein

Expression

system

sde

velopm

ent

Full-leng

thprotein

Mitraan

dZh

ang(199

4)N.taba

cum

L.Cellsuspe

nsioncultu

rehLf,0.7–

2.7%

oftotalsolub

leprotein

Expression

system

sde

velopm

ent

Part-le

ngth

(48kD

a)protein

Cho

ietal.(200

3)

N.taba

cum

L.Plan

thLf,0.1–

0.3%

oftotalsolub

leprotein

Expression

system

sde

velopm

ent

Full-leng

thprotein

Salm

onet

al.(199

8)

N.taba

cum

L.Plan

thLf,0.1–

0.8%

oftotalsolub

leprotein

Resistan

ceto

Ralstonia

solana

cearum

Full-leng

th,pa

rt-le

ngth

(48kD

a)protein

Zhan

get

al.(199

8b)

N.taba

cum

L.cv.Xan

thi

Plan

tbLf,da

tano

tshow

nRe

sistan

ceto

Rhizoctoniasolani

Full-leng

thprotein

Ngu

yenet

al.(201

1)To

bacco(N.be

ntha

miana

Dom

in.)

Plan

thLfN,0.6%

oftotalsolub

leprotein

Expression

system

sde

velopm

ent

Full-leng

thhLfN

protein

Liet

al.(200

4)

Potato

(Solan

umtube

rosum

L.)

Plan

thLf,0.01

–0.1%

oftotalsolub

leprotein

Food

additives

Full-leng

thprotein

Cho

ngan

dLang

ridge

(200

0)To

mato(Lycop

ersicon

esculentum

Mill.)

Plan

thLf,da

tano

tshow

nBa

cterialw

iltresistan

ceFull-leng

thLeeet

al.(200

2)

Rice

(Oryza

sativaL.)

Plan

thLf,0.5–

5.0g/kg

ofde

husked

rice

grains

Pharmaceu

ticals

Full-leng

thprotein

Nan

diet

al.(200

2)

O.sativaL.

Cellculture

rhLf,2–

4%of

thetotalsolub

leprotein

Food

additives

Full-leng

thprotein

Suzuki

etal.(200

3)

O.sativaL.

Plan

thLf,2.0mg/gof

dehu

sked

seed

sPh

armaceu

ticals

Full-leng

thprotein

Rachmaw

atie

tal.(200

5)O.sativaL.

Plan

tpLf,0.1%

ofricebran

weigh

tFood

additives

Full-leng

thprotein

Leeet

al.(201

0)O.sativaL.

Plan

thLf,0.45

%of

totald

ryweigh

tDevelop

men

tof

selectionsystem

Full-leng

thprotein

Linet

al.(201

0)Ginseng

(Pan

axginsen

gC.A.

Meyer)

Cellsuspe

nsioncultu

rehLf,3%

oftotalsolub

leprotein

Expression

system

sde

velopm

ent

Full-leng

th,pa

rt-le

ngth

(40kD

a)protein

Kwon

etal.(200

3)

Pear

(Pyrus

sp.)

Plan

tbLf,da

tano

tshow

nErwinia

amylovoraresistan

ceFull-leng

th,pa

rt-le

ngth

(60kD

a)Malno

yet

al.(200

3)

continued

A.I. Yemets et al. Plant-based biopharming of recombinant human Lf

995Cell Biol Int 38 (2014) 989–1002 © 2014 International Federation for Cell Biology

activity. Only two bioreactors – O. sativa and A. niger – arealready in commercial use; hence, the search for alternativehosts for rhLf expression remains attractive forbiotechnologists.

Lactoferrin expression as a tool for theenhancement of non-specific plant pathogenresistance

In contrast to mammals, there is no mobile defensive cellsand adaptive somatic immune system in plants (Joet al., 2006; Jones and Dangl, 2006). Instead, they rely onthe innate immunity of single cell and on systemic signalsemanating from infection sites (Jo et al., 2006; Jones andDangl, 2006).

Due to multiple protective Lf properties, its genes aredesirable candidates for the introduction into the genomesof economically important higher plant species for theenhancement of their immune response. Several attempts tostrengthen plant pathogen resistance have been done by thetransfer of hLf gene into tobacco (Zhang et al., 1998b;Nguyen et al., 2011), tomato (Lee et al., 2002), pear (Malnoyet al., 2003), rice (Takes et al., 2005) and wheat (Hanet al., 2012). As a result, primary lines of the transformedtobacco plants (T0) with different resistance to untimely wiltcaused by the bacteria Ralstonia solanacearum wereobtained, and the plants of XNC/hlf-T1-2 and XNC/hlf-T1-8 transgenic lines showing the highest resistance couldproduce normal seeds similar to wild-type plants (Zhanget al., 1998b). Quantification of total Lf confirmed a positivecorrelation 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 low-molecular weight together with the full-length (80 kDa) Lfwas also found in the highly resistant transgenic tobacco cellsexpressing hLf (Mitra and Zhang, 1994). A certain amountof Lf could be truncated or degraded during the transfor-mation process (Zhang et al., 1998b).

Besides transgenic tobacco, resistance to R. solanacearumwas achieved in susceptible tomato L. esculentum cultivarF7926-96 expressing the full-length hLf gene under thecontrol of 35S promoter (Lee et al., 2002). First transgenic(T1) generation of Lf-expressing tomato plants possessedbacterial wilt resistance on the early stages of infection (Leeet al., 2002). However, the level of resistance strongly variedamong the individual tomato transformed plants that alsomay occur due to Lf differential expression (Lee et al. 2002)as compared to earlier results (Zhang et al., 1998b). The non-transgenic plants inoculated with R. solanacearum suspen-sion (1� 107–108CFU/mL) totally wilted on 10–11th dayafter the inoculation, while the wilting of LF-T1-29 andLF-T1-34 Lf-expressing lines was significantly delayed to 23–24th day. LF-T1-34 transgenic plants wilted 16 days later

Table

1.(Continued

)

Species

Type

ofcultu

reLactoferrin

origin,expression

level

Purposeof

use

Details

References

protein

Sweetpo

tato

(Ipom

oeaba

tatas

(L.)Lam.)

Cellsuspe

nsioncultu

rehLf,0.32

%of

totalsolub

leprotein

Expression

system

sde

velopm

ent

Full-leng

thprotein

Min

etal.(200

6)

Sibe

rianginsen

g(Acantho

pana

xsenticosus

(Rup

r.&Maxim

)Harms.)

Cellsuspe

nsioncultu

rehLf,3.6%

oftotalsolub

leprotein

Expression

system

sde

velopm

ent

Full-leng

th(80kD

a),pa

rt-le

ngth

(35kD

a)protein

Joet

al.(200

6)

Barle

y(Horde

umvulgareL.)

Plan

thLf,3ng

/mgof

totalsolub

leprotein

Expression

system

sde

velopm

ent

Full-leng

thprotein

Kam

enarovaet

al.(200

7)

H.vulgareL.

Plan

thLf

Expression

system

sde

velopm

ent

mRN

ATana

sien

koet

al.(201

1)Arabido

psis(Arabido

psisthaliana

(L.)Heynh

.)Plan

tbLf,da

tano

tshow

nFung

alinfectionresistan

ceFull-leng

thprotein

Ngu

yenet

al.(201

1)

Alfa

lfa(M

edicag

osativaL.)

Plan

thLf,0.00

47%

oftotalsolub

leprotein

Pseu

domon

assyrin

gaepv.

syrin

gaean

dClaviba

cter

michiga

nensisresistan

ce

Full-leng

thprotein

Stefan

ovaet

al.(201

2)

Whe

at(Triticum

aestivum

L.)

Plan

tbLf,21

–67

ng/m

gof

leaf

tissue

Fusariu

mgram

inearum

resistan

ceFull-leng

thprotein

Han

etal.(201

2)

Plant-based biopharming of recombinant human Lf A.I. Yemets et al.

996 Cell Biol Int 38 (2014) 989–1002 © 2014 International Federation for Cell Biology

compared to control, whereas wilting of LF-T1-11 and LF-T1-28 Lf-expressing tomato lines was delayed just for 4 days.Nevertheless, 44–55% of T2 generation maintained wiltingresistance to the fruit ripening stage after the bacterialinoculation. Therefore, regardless of Lf resistance instability,transformation of the susceptible tomato cultivars by hLfgene gave substantial benefits to transgenic plants incomparison to non-transformed ones. Several edible andcommercial plant species were transformed also by bovine Lfgene (bLf). For instance, the transformation of pear plants bybLf conferred the resistance to fire blight caused by bacteriaErwinia amylovora (Malnoy et al., 2003). In this investiga-tion, nine transgenic clones were obtained, but only six ofthem underwent the detailed analysis of transgene expres-sion and disease resistance as the remaining clones expressedbLf. However, in vitro cultivated plants had higher Lftranscript levels than acclimatized plants. Despite this, allclones produced the expected Lf protein in vivo. In alltransgenic lines the reduction of infection symptoms ofshoots in vitro was 17%, rooted plants 30%, and graftedplants 60%. Furthermore, increased in vitro resistance of thetransformed pear plants to other diseases – Agrobacteriumtumefaciens-induced crown galls and Pseudomonas syrin-gae-caused bacterial blast – was achieved.

Lf-expressing rice plants were examined for diseaseresistance 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 expressionof hLf and hLfN (the N-lobe of hLf) were tested. This N-terminal protein domain could be released by pepsindigestion of Lf as an antibacterial peptide called lactoferricin(Bellamy et al., 1992), which proved to be more potent abactericidal agent than Lf itself (Tomita et al., 1991). BothhLf- and hLfN-transformed rice plants produced up to0.1mg of hLf/hLfN protein per 1 g of leaves. Because of thesignificant difference in molecular weights of plant-derivedfull-length Lf andmilk hLf, the comparative analysis of theseproteins was performed. It was shown that the rice-derivedhLf contains plant-type oligosaccharide chains, which maypartially account for the difference in milk hLf and rice hLfmolecular weights. The antibacterial resistance assay showedthe resistance of hLf- and hLfN-transformed rice plants to B.plantarii, which causes blight in the seedlings. Some hLf-transformed rice plants infected with Rice dwarf virusshowed a delay in symptoms manifestation; however, thisobservation could be explained not with the enhancement ofhLf-transformants resistance, but with the delay of theirgrowth.

The retardation of Lf-expressing tobacco and pear growthas compared to non-transformed plants was shownpreviously (Zhang et al., 1998b; Malnoy et al., 2003). Thereduction of the total pear tree height was ~30% (Malnoy

et al., 2003), the hLf-expressing tobacco plants ~10% (Zhanget al., 1998b) and the hLf-expressing rice plants 10–20%(Takes et al., 2005). In turn, no direct or indirect effects of Lfor its fragments were found in vitro and in vivo on O. sativaresistance to the rice blast disease caused by P. oryzae (Takeset al., 2005). As for the fungal infection, the transgenicN. tabacum var Xanthi andA. thaliana plants expressing bLfunder 35S promoter showed resistance against fungalpathogen Rhizoctonia solani, the causal agent of economi-cally unfavorable damping-off plant diseases (Nguyenet al., 2011). The transgenic plants are characterized bythe presence of 77 kDa Lf protein corresponding to themolecular weight of full-length protein. Tobacco leafbioassay also showed plant resistance to the fungal pathogen.As for Arabidopsis, the T2 transgenic generation seeds weregerminated in pots containing R. solani cornmeal mycelialinoculums. Most seedlings in the control pots died out soonafter the germination, while the majority of the transgenicseedlings grew normally, which indicates the successful gainof resistance against R. solani-induced damping-off. Finally,R. solani resistance was confirmed in all bLf-expressingArabidopsis and tobacco lines (Nguyen et al., 2011).

A bLf has also been introduced into a wheat cultivar,Bobwhite, susceptible to fusarium head blight (FHB) causedby the fungus Fusarium graminearum (Han et al., 2012). Thelevel of full-length target protein in T8 progeny of transgeniclines varied from 21 to 67 ng/mg of leaf tissue. It was foundthat Lf inhibits the growth of fungi in transgenic plantsboth in vitro and in vivo. Variation in the resistance levelsamong the independent transformation events correlatedwith the actual amount of Lf in the transgenic lines, whichcorroborates earlier results (Malnoy et al., 2003). The glumeswere infected during the manual inoculation of wheatinflorescence with a spore suspension, which could beexplained by the difference in concentrations of bLf in theglumes and leaves (0.11% and 0.52% of the total solubleprotein, respectively). Nevertheless, all transgenic linesshowed a significant level of the resistance as comparedto the untransformed Bobwhite, susceptible (Wheaton)and resistant (ND 2710) to FHB wheat lines (Han et al.,2012).

Alfalfa plants expressing hLf displayed mild symptoms ofPseudomonas syringae pv. syringae infection in comparisonwith non-transformed plants (Stefanova et al., 2012).Moreover, the inoculation of fresh alfalfa branches withClavibacter michiganensis caused different level of symp-toms reduction (Stefanova et al., 2012) that could beexplained by different Lf content.

Eventually, the transformation of some higher plantspecies by a set of Lf genes (bLf, hLf, and hLfN) confersbroad-spectrum resistance against viral, bacterial, and fungalplant diseases (Zhang et al., 1998b; Liu et al., 1999; Leeet al., 2002; Takes et al., 2005) and is a promising tool for the

A.I. Yemets et al. Plant-based biopharming of recombinant human Lf

997Cell Biol Int 38 (2014) 989–1002 © 2014 International Federation for Cell Biology

enhancement of plant biotic stress resistance crucial for foodproduction safety.

Important aspects of biosafety in lactoferrin-producing plants

The production of heterologous proteins in plant-basedexpression systems also known as molecular farminginvolves the use of genetically modified (GM) plants thatare obliged to undergo health and environmental risksassessment (Ma et al., 2003; Breyer et al., 2009). Biologicalsafety of GM plants can be achieved by the application ofnew vector constructs carrying health- and environmental-ly-friendly selective marker genes based exclusively ongenetic information already present in host plant, forinstance, mutant a-tubulin gene instead of widespreadantibiotic resistance genes (Blume et al., 2008; Yemetset al., 2008). Recently, hLf-expressing transgenic barley wasobtained via biolistic transformation using plasmid vectorcarrying this mutant a-tubulin gene providing resistance todinitroaniline herbicides (Tanasienko et al., 2011).

The main environmental risks remain foreign gene flowfrom pollen/seed dispersal, horizontal gene transfer, as wellas potentially toxic effects of recombinant proteins onsymbiotic microorganisms, pollinating insects and herbi-vores (Ma et al., 2003). Hence, field growing of GM plants or“the usage of such plants outside the constraints of physicalcontainment” has to be authorized by Animal and PlantHealth Inspection Service (APHIS) in the USA (Breyeret al., 2009). As for Lf production, only rice-based expressionsystem (Ventria Bioscience, USA) has been authorized byAPHIS for the treatment of iron deficiency and acutediarrhea (Bethell and Huang, 2004). For the appropriateauthorization also Standard Operating Procedures (SOPs,USA) with the detailed instructions and additional guidancefor field tests exists (USDA, 2005 Environmental Assess-ment). According to this document, rhLf synthesized inO. sativa ssp. japonica cv. Taipei 309 is allowed to becultivated, harvested, stored and transported only after thephysical separation from the conventional rice crops byequipment not used for the production or storage of acommercial food rice. Moreover, the rice-derived rhLf hasbeen marked as Generally Recognized as Safe (GRAS), thatmeans its intentional addition to food after subjecting topremarket review and approval of qualified experts of Foodand Drug Administration (FDA) as having been shownadequately to be safe under the conditions of its intended use(Bethell, 2004). Since transgenic rice was used for large-scalehLf production, one of the major environmental safetyconcerns is the possibility of transgenic hLf-expressing riceescaping into the environment (Lin et al., 2010). A newstrategy for controlling the spread of hLf-expressing rice wasproposed based on the use of RNA interference cassette that

suppresses the expression of rice enzyme able to detoxify theherbicide bentazon tagged with gene of interest. Differentcassettes carrying the desirable traits (hLf expression,enhancement of bentazon sensitivity and glyphosate resis-tance for the transformants selection) were located in asingle T-DNA fragment that allows separating of one or twotrait(s) during the transformation. To test this, bothhomozygous and heterozygous transgenic plants weretreated by 1000mg/L bentazon, half its recommendeddose for rice weed control. The sensitivity of all plantsto bentazon treatment suggested the inheritance of allthree traits in case of transgenic and nontransgenic ricehybridization (Lin et al., 2010). Thus the approach describedcould potentiate the separation of Lf-expressing GMplants in the environment and, therefore, decrease ofenvironmental risks.

Conclusions and future prospects

As natural sources of pharmaceutical proteins and otherbioactive molecules are rather limited, there is a growinginterest to the development of novel bioreactors for theproduction of pharmaceuticals. Plant-based expressionsystems have a set of advantages such as sufficientpolypeptides expression, eukaryotic protein folding andrelatively simple process of target protein purification incomparison with yeast and fungal systems, allowing thedevelopment of highly efficient technologies for the large-scale production of Lf. Among all engineered plant-systemsfor Lf synthesis the most promising ones are based onrice. Microalgae (Franklin and Mayfield, 2005), barley(Kamenarova et al., 2007; Tanasienko et al., 2009, 2011;Yemets et al., 2012) and alfalfa (Stefanova et al., 2013) couldalso be used as alternative platforms for the accumulation ofhigh levels of the recombinant proteins.

Acknowledgement

We appreciate suggestions from Andrej. S. Mosyakin(Institute of Botany, National Academy of Sciences ofUkraine, Kiev) about the improvement of manuscriptlanguage.

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