Documente Academic
Documente Profesional
Documente Cultură
doi: 10.1002/cbin.10304
REVIEW
Abstract
Recombinant proteins are currently recognized as pharmaceuticals, enzymes, food constituents, nutritional additives,
antibodies and other valuable products for industry, healthcare, research, and everyday life. Lactoferrin (Lf), one of the
promising human milk proteins, occupies the expanding biotechnological food market niche due to its important versatile
properties. Lf shows antiviral, antimicrobial, antiprotozoal and antioxidant activities, modulates cell growth rate, binds
glycosaminoglycans and lipopolysaccharides, and also inputs into the innate/specic immune responses. Development of
highly efcient human recombinant Lf expression systems employing yeasts, lamentous fungi and undoubtedly higher
plants as bioreactors for the large-scale Lf production is a biotechnological challenge. This review highlights the advantages
and disadvantages of the existing non-animal Lf expression systems from the standpoint of protein yield and its biological
activity. Special emphasis is put on the benets of monocot plant system for Lf expression and the biosafety aspects of the
transgenic Lf-expressing plants.
Keywords: human lactoferrin; recombinant expression systems; plant-based biofarming; plant-pathogen resistance;
biosafety
Introduction
Recently numerous recombinant proteins have been used
intensely in pharmacy, industry and research and, therefore,
have to meet a range of sophisticated quality requirements,
before they could be considered safe, in particular, an
extra high-purity (Ma et al., 2003). According to Good
Manufacturing Practice, all recombinant proteins must be
sufciently pure and homogeneous with contaminants
removed to acceptable levels (Fischer et al., 2012). It is
important either to improve the protein production from
their native sources or to search for new ones together with
the development of the efcient protein expression systems
and the advance of protein extraction protocols.
Novel recombinant proteins, also referred to as highmolecular drugs, could be the targeted agents for the
treatment of such common health problems of industrial
countries as oncological, cardiovascular and infectious
diseasesall critical to an expanding and aging human
population (Elbehri, 2005). The existing pharmaceutical
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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 4.55.5 g/kg of
the selected O. sativa homozygous line (LF164) over nine
generations (Nandi et al., 2005). As a result, quantitative
analysis of this transgenic line indicated 0.5% rhLf in brown
rice our amounting to >25% of the total soluble protein.
Comparable data were reported by Lin et al. (2010), where
the concentration of hLf expressed in seeds under the
glutelin (Gtl) promoter reached 0.45% of the total dry weight
of the dehusked rice seeds. The N-terminal sequence of this
rice-derived hLf was identical to that of native hLf mature
protein (Lin et al., 2010) as in a previous study (Nandi
et al., 2002). A similar approach with codon optimization
and Gt1 promoter resulted in 93130 mg of total rhLf in the
transformed rice seeds, which was patented by Huang et al.
(2010). The comparably high rhLf expression level achieved
in rice grains provides a strong base for the development of
its low-cost downstream processing (Nandi et al., 2005).
For the investigation of other rice expression systems,
a range of different regulatory sequences were tested
(Rachmawati et al., 2005). Thus, the inuence of the
constitutive maize ubiquitin-1 promoter, sequence encoding
native hLf or rice glutelin signal peptides was examined on
the hLf expression in Javanica rice. The expression level of
hLf in the vegetative tissue of the transgenic plant was <0.8%
of total soluble protein, but it was signicantly higher in
seeds. The highest level of rhLf expression with rice glutelin
signal peptide reached 2.0 mg/g in dehusked seeds, while
recombinant hLf expression level with hLf signal peptide was
only 1.6 mg/g. Therefore, the use of the correct signal peptide
would be important to locate the target protein to the
appropriate protein body of endosperm transgenic rice seeds
that protects Lf from the protease digestion. The presence of
recombinant hLf in all parts of transgenic plants conrmed
the constitutivity of maize ubiquitin-1 promoter. Low
recombinant hLf expression level in vegetative part of rice
plants may be explained by the enhanced protein susceptibility to protease degradation. The level of the recombinant
hLf production in transgenic seeds reached 15% of the total
soluble protein compared to tissue-specic Gtl promoter use
(20%) (Nandi et al., 2002; Lin et al., 2010). Nevertheless, Nterminal amino acid of hLf was identical to that of milk hLf
(Rachmawati et al., 2005). Therefore, such an approach gives
an opportunity to use Lf expression not only for the
accumulation and further oral administration as food
additives or pharmaceuticals, but also for plant protection
against numerous pathogens (Nandi et al., 2002).
Another promoter was chosen to produce the porcine
full-length recombinant Lf (prLf) in Japonica rice cultivar
TNG67 (Lee et al., 2010). The expression level of porcine
rLf in rice bran under the rice actin promoter was estimated
994
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O. sativa L.
O. sativa L.
O. sativa L.
Ginseng (Panax ginseng C.A.
Meyer)
Pear (Pyrus sp.)
O. sativa L.
Tomato (Lycopersicon
esculentum Mill.)
Rice (Oryza sativa L.)
Plant
Plant
Plant
Plant
Cell suspension culture
Plant
Cell culture
Plant
Plant
Plant
Plant
Plant
N. tabacum L.
hLf, 2 g/L
hLf, 25 mg/L
hLf, 25 mg/L
Mycelia
Mycelia
N. tabacum L.
A. awamori
Higher plants
Tobacco (Nicotiana tabacum L.)
N. tabacum L.
A. nidulans
Mycelia
Pichia methanolica
Filamentous fungi
Aspergillus oryzae
Type of culture
Pichia pastoris
Yeast
Saccharomyces cerevisiae
Species
Full-length protein
Full-length protein
Details
Full-length protein
Full-length protein
Full-length protein
Pharmaceuticals
Food additives
Development of selection system
Expression systems development
Food additives
Pharmaceuticals
Food additives
Full-length protein
Full-length protein
Full-length protein
Full-length, part-length (40 kDa)
protein
Full-length, part-length (60 kDa)
Full-length protein
Full-length protein
Full-length
Full-length protein
Resistance to Ralstonia
Full-length, part-length (48 kDa)
solanacearum
protein
Resistance to Rhizoctonia solani Full-length protein
Expression systems development Full-length hLfN protein
Pharmaceuticals
Pharmaceuticals
Pharmaceuticals
Pharmaceuticals
Pharmaceuticals
Purpose of use
continued
References
995
996
Plant
Full-length protein
Pseudomonas syringae pv.
syringae and Clavibacter
michiganensis resistance
Fusarium graminearum resistance Full-length protein
H. vulgare L.
Plant
Arabidopsis (Arabidopsis thaliana Plant
(L.) Heynh.)
Alfalfa (Medicago sativa L.)
Plant
Jo et al. (2006)
Expression systems development Full-length (80 kDa), part-length
(35 kDa) protein
hLf, 3.6% of total soluble protein
Species
Table 1. (Continued)
Type of culture
Purpose of use
Details
References
compared to control, whereas wilting of LF-T1-11 and LFT1-28 Lf-expressing tomato lines was delayed just for 4 days.
Nevertheless, 4455% of T2 generation maintained wilting
resistance to the fruit ripening stage after the bacterial
inoculation. Therefore, regardless of Lf resistance instability,
transformation of the susceptible tomato cultivars by hLf
gene gave substantial benets to transgenic plants in
comparison to non-transformed ones. Several edible and
commercial plant species were transformed also by bovine Lf
gene (bLf). For instance, the transformation of pear plants by
bLf conferred the resistance to re blight caused by bacteria
Erwinia amylovora (Malnoy et al., 2003). In this investigation, nine transgenic clones were obtained, but only six of
them underwent the detailed analysis of transgene expression and disease resistance as the remaining clones expressed
bLf. However, in vitro cultivated plants had higher Lf
transcript levels than acclimatized plants. Despite this, all
clones produced the expected Lf protein in vivo. In all
transgenic lines the reduction of infection symptoms of
shoots in vitro was 17%, rooted plants 30%, and grafted
plants 60%. Furthermore, increased in vitro resistance of the
transformed pear plants to other diseases Agrobacterium
tumefaciens-induced crown galls and Pseudomonas syringae-caused bacterial blast was achieved.
Lf-expressing rice plants were examined for disease
resistance in vitro and in vivo against three pathogens:
Rice dwarf virus, bacterium Burkholderia (Pseudomonas)
plantarii, and fungus Pyricularia oryzae (Takase et al., 2005).
Two transgenic O. sativa lines with constitutive expression
of hLf and hLfN (the N-lobe of hLf) were tested. This Nterminal protein domain could be released by pepsin
digestion of Lf as an antibacterial peptide called lactoferricin
(Bellamy et al., 1992), which proved to be more potent a
bactericidal agent than Lf itself (Tomita et al., 1991). Both
hLf- and hLfN-transformed rice plants produced up to
0.1 mg of hLf/hLfN protein per 1 g of leaves. Because of the
signicant difference in molecular weights of plant-derived
full-length Lf and milk hLf, the comparative analysis of these
proteins was performed. It was shown that the rice-derived
hLf contains plant-type oligosaccharide chains, which may
partially account for the difference in milk hLf and rice hLf
molecular weights. The antibacterial resistance assay showed
the resistance of hLf- and hLfN-transformed rice plants to B.
plantarii, which causes blight in the seedlings. Some hLftransformed rice plants infected with Rice dwarf virus
showed a delay in symptoms manifestation; however, this
observation could be explained not with the enhancement of
hLf-transformants resistance, but with the delay of their
growth.
The retardation of Lf-expressing tobacco and pear growth
as compared to non-transformed plants was shown
previously (Zhang et al., 1998b; Malnoy et al., 2003). The
reduction of the total pear tree height was ~30% (Malnoy
Cell Biol Int 38 (2014) 9891002 2014 International Federation for Cell Biology
997
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Received 3 October 2013; accepted 31 March 2014.
Final version published online 19 May 2014.
Cell Biol Int 38 (2014) 9891002 2014 International Federation for Cell Biology