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/specific immune responses. Development of highly efficient human recombinant Lf expression systems employing yeasts, filamentous 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 benefits 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 sufficiently 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 efficient 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 diseases—all 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 modifications (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, unprofitable purification 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 sufficient 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: [email protected]
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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 the conventional “first 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 fluids (milk, colostrum, saliva, tears, etc.) (Adlerova et al., 2008; GonzálezChávez et al., 2009; Lönnerdal 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 (Lönnerdal 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 (García-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; 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 membrane proteoglycan layer to lysozyme activity (Ellison and Giehl, 1991; Lönnerdal and Suzuki, 2013). Lf has antiviral activity against a broad range of RNA and DNA-containing human and animal viruses (GarcíaMontoya et al., 2012; Lönnerdal and Suzuki, 2013) by inhibiting virus–host interaction, virus trafficking or direct binding of the viral particle by the blocking of glycosaminoglycan viral receptors, especially heparan sulfate (GarcíaMontoya et al., 2012). It acts strongly against HIV in vitro (García-Montoya et al., 2012) by inhibiting viral replication inside the host cell (Swart et al., 1996; Qiu et al., 1998; García-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 Fe3þ sequestration (Zarember et al., 2007; González-Chávez et al., 2009). Thus, Lf eliminates Candida albicans and C. krusei by the alterating the permeability of their cell surfaces (Wakabayashi et al., 1996; García-Montoya et al., 2012). Many reports indicate the beneficial 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 (García-Montoya et al., 2012). It can promote apoptosis and arrest tumor growth in vitro (Lönnerdal and Suzuki, 2013). Among the other factors associated with Lf’s 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 Crohn’s disease (Vogel, 2012). Though Lf levels are increased in the synovial fluid of the inflamed knee joints, suggesting neutrophil infiltration, 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; Lönnerdal and Suzuki, 2013), suggesting an important role of Lf in fat metabolism due to its antiadipogenic, antioxidative and anti-inflammatory activities (Lönnerdal and Suzuki, 2013). Despite numerous beneficial 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); 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); 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-specific 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 purification 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 purification procedures are required. For this reason, such widely used producers as yeast, filamentous fungi, and higher plants are being considered as the most efficient 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
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combination and also the high cell culture density, make P. pastoris an efficient 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 identification of the optimal one that facilitates the translocation of rhLf into the secretory pathway and finally into the culture medium (Choi et al., 2008). From all tested S. cerevesiae signal sequences, alpha mating factor prepro has been selected and modified 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 confirmed 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 1.5–2 mg/L (Liang and Richardson, 1993). However, the significant disadvantage of yeasts as heterologous protein bioreactors is their inability to provide proper eukaryotic protein post-translational modifications as amidation and prolylhydroxylation, and also some types of phosphorylation and glycosylation (Cregg and Higgins, 1995). In turn, the secretion potential of filamentous 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 modifications of polypeptides as N-glycosylation and disulfide bonds formation (Maras et al., 1999). Lf expression in three filamentous fungi: A. oryzae under the control of a-amylase promoter and the 30 -flanking 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 purified from growth medium of A. oryzae using CM Sephadex C50, SDS/PAGE silver staining and Lf purified 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, filamentous 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 modifications required for a full-fledged protein activity, as well as the protection of polypeptides from the proteolytic degradation (Franklin and Mayfield, 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 efficient against such bacterial phytopathogens as Xanthomonas campestris pv. phaseoli, Pseudomonas syringae pv. phaseolicola, P. syringae pv. syringae and Clavibacter flaccumfaciens pv. flaccumfaciens 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 efficiency 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 efficient 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 efficient system for rhLf production is alfalfa, characterized
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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 confirmation 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 (Lönnerdal, 2002; Fischer et al., 2004; Stefanova et al., 2008). Production of foreign proteins in plant cell cultures may be more beneficiary than in whole plants, because of time constraints, better control and reproducible conditions in bioreactors as compared to field-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).
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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). Purified 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 5–10 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 first 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 significantly enhance the ultimate yield of target peptides. Additionally, the promoter shows specifically 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 benefits 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 a2–6-linked neuraminic acid, b1–4-linked galactose and a1–6-linked fucose glycans – typical for mammals (Spik et al., 1982), while rhLf from “koshihikari” rice cultivar has a1–3-linked fucose and b1–2-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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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 4.5–5.5 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 flour 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 93–130 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 influence 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
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/specific immune responses. Development of highly efficient human recombinant Lf expression systems employing yeasts, filamentous 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 benefits 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 sufficiently 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 efficient 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 diseases—all 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 modifications (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, unprofitable purification 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 sufficient 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: [email protected]
Cell Biol Int 38 (2014) 989–1002 © 2014 International Federation for Cell Biology
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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 the conventional “first 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 fluids (milk, colostrum, saliva, tears, etc.) (Adlerova et al., 2008; GonzálezChávez et al., 2009; Lönnerdal 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 (Lönnerdal 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 (García-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; 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 membrane proteoglycan layer to lysozyme activity (Ellison and Giehl, 1991; Lönnerdal and Suzuki, 2013). Lf has antiviral activity against a broad range of RNA and DNA-containing human and animal viruses (GarcíaMontoya et al., 2012; Lönnerdal and Suzuki, 2013) by inhibiting virus–host interaction, virus trafficking or direct binding of the viral particle by the blocking of glycosaminoglycan viral receptors, especially heparan sulfate (GarcíaMontoya et al., 2012). It acts strongly against HIV in vitro (García-Montoya et al., 2012) by inhibiting viral replication inside the host cell (Swart et al., 1996; Qiu et al., 1998; García-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 Fe3þ sequestration (Zarember et al., 2007; González-Chávez et al., 2009). Thus, Lf eliminates Candida albicans and C. krusei by the alterating the permeability of their cell surfaces (Wakabayashi et al., 1996; García-Montoya et al., 2012). Many reports indicate the beneficial 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 (García-Montoya et al., 2012). It can promote apoptosis and arrest tumor growth in vitro (Lönnerdal and Suzuki, 2013). Among the other factors associated with Lf’s 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 Crohn’s disease (Vogel, 2012). Though Lf levels are increased in the synovial fluid of the inflamed knee joints, suggesting neutrophil infiltration, 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; Lönnerdal and Suzuki, 2013), suggesting an important role of Lf in fat metabolism due to its antiadipogenic, antioxidative and anti-inflammatory activities (Lönnerdal and Suzuki, 2013). Despite numerous beneficial 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); 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); 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-specific 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 purification 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 purification procedures are required. For this reason, such widely used producers as yeast, filamentous fungi, and higher plants are being considered as the most efficient 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
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combination and also the high cell culture density, make P. pastoris an efficient 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 identification of the optimal one that facilitates the translocation of rhLf into the secretory pathway and finally into the culture medium (Choi et al., 2008). From all tested S. cerevesiae signal sequences, alpha mating factor prepro has been selected and modified 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 confirmed 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 1.5–2 mg/L (Liang and Richardson, 1993). However, the significant disadvantage of yeasts as heterologous protein bioreactors is their inability to provide proper eukaryotic protein post-translational modifications as amidation and prolylhydroxylation, and also some types of phosphorylation and glycosylation (Cregg and Higgins, 1995). In turn, the secretion potential of filamentous 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 modifications of polypeptides as N-glycosylation and disulfide bonds formation (Maras et al., 1999). Lf expression in three filamentous fungi: A. oryzae under the control of a-amylase promoter and the 30 -flanking 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 purified from growth medium of A. oryzae using CM Sephadex C50, SDS/PAGE silver staining and Lf purified 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, filamentous 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 modifications required for a full-fledged protein activity, as well as the protection of polypeptides from the proteolytic degradation (Franklin and Mayfield, 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 efficient against such bacterial phytopathogens as Xanthomonas campestris pv. phaseoli, Pseudomonas syringae pv. phaseolicola, P. syringae pv. syringae and Clavibacter flaccumfaciens pv. flaccumfaciens 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 efficiency 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 efficient 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 efficient system for rhLf production is alfalfa, characterized
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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 confirmation 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 (Lönnerdal, 2002; Fischer et al., 2004; Stefanova et al., 2008). Production of foreign proteins in plant cell cultures may be more beneficiary than in whole plants, because of time constraints, better control and reproducible conditions in bioreactors as compared to field-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).
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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). Purified 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 5–10 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 first 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 significantly enhance the ultimate yield of target peptides. Additionally, the promoter shows specifically 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 benefits 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 a2–6-linked neuraminic acid, b1–4-linked galactose and a1–6-linked fucose glycans – typical for mammals (Spik et al., 1982), while rhLf from “koshihikari” rice cultivar has a1–3-linked fucose and b1–2-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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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 4.5–5.5 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 flour 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 93–130 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 influence 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
