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International Journal of Antimicrobial Agents 33 (2009) 301.e1–301.e8
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International Journal of Antimicrobial Agents
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Review
Lactoferrin: structure, function and applications
Susana A. González-Chávez, Sigifredo Arévalo-Gallegos, Quintín Rascón-Cruz ∗
Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Cd. Universitaria s/n, CP 31170, Chihuahua, Chihuahua, Mexico
a r t i c l e
i n f o
a b s t r a c t
Keywords:
Lactoferrin
Iron-binding protein
Transferrin
Functional protein
Lactoferrin (LF) is an 80 kDa iron-binding glycoprotein of the transferrin family that is expressed in most
biological fluids and is a major component of the mammalian innate immune system. Its protective effects
range from direct antimicrobial activities against a large panel of microorganisms, including bacteria,
viruses, fungi and parasites, to anti-inflammatory and anticancer activities. These extensive activities are
made possible by mechanisms of action utilising not only the capacity of LF to bind iron but also interactions of LF with molecular and cellular components of both host and pathogens. This review summarises
the putative antimicrobial mechanisms, clinical applications and heterologous expression models for LF.
© 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction
due to two mechanisms. The first is iron sequestration in sites
of infection, which deprives the microorganism of this nutrient,
thus creating a bacteriostatic effect. The other mechanism is the
direct interaction of the LF molecule with the infectious agent.
The positive amino acids in LF can interact with anionic molecules
on some bacterial, viral, fungal and parasite surfaces, causing cell
lysis.
Considering the physiological capabilities of LF in host defence,
in addition to current pharmaceutical and nutritional needs, LF is
considered to be a nutraceutical and for several decades investigators have searched for the most convenient way to produce it.
Today, we can obtain it as native LF isolated mostly from the milk
and colostrum of several mammals, or as recombinant LF (rLF)
generated from bacterial, fungal and viral expression systems. The
expression of this protein has also been attained in higher organisms such as plants and mammals.
Lactoferrin (LF) is a non-haem iron-binding protein that is part
of the transferrin protein family, along with serum transferrin,
ovotransferrin, melanotransferrin and the inhibitor of carbonic
anhydrase [1], whose function is to transport iron in blood serum.
LF is produced by mucosal epithelial cells in various mammalian
species, including humans, cows, goats, horses, dogs and several
rodents. Recent studies have shown that LF is also produced by fish,
as it has been identified in rainbow trout eggs using molecular biology techniques [2]. This glycoprotein is found in mucosal secretions,
including tears, saliva, vaginal fluids, semen [3], nasal and bronchial
secretions, bile, gastrointestinal fluids, urine [4] and most highly in
milk and colostrum (7 g/L) [5], making it the second most abundant protein in milk [6], after caseins. It can also be found in bodily
fluids such as blood plasma and amniotic fluid. LF is also found in
considerable amounts in secondary neutrophil granules (15 ␮g/106
neutrophils) [7], where it plays a significant physiological role. LF
possesses a greater iron-binding affinity and is the only transferrin with the ability to retain this metal over a wide pH range [8],
including extremely acidic pH. It also exhibits a greater resistance to
proteolysis. In addition to these differences, LF’s net positive charge
and its distribution in various tissues make it a multifunctional
protein. It is involved in several physiological functions, including: regulation of iron absorption in the bowel; immune response;
antioxidant, anticarcinogenic and anti-inflammatory properties;
and protection against microbial infection, which is the most widely
studied function to date. The antimicrobial activity of LF is mostly
∗ Corresponding author. Tel.: +52 614 414 4492.
E-mail address: [email protected] (Q. Rascón-Cruz).
2. Structure and properties
LF (Fig. 1) is an 80 kDa glycosylated protein of ca. 700 amino acids
with high homology among species. It is a simple polypeptide chain
folded into two symmetrical lobes (N and C lobes), which are highly
homologous with one another (33–41% homology). These two lobes
are connected by a hinge region containing parts of an ␣-helix
between amino acids 333 and 343 in human LF (hLF) [1], which
provides additional flexibility to the molecule [3]. The polypeptide
chain includes amino acids 1–332 for the N lobe and 344–703 for
the C lobe and is made up of ␣-helix and ␤-pleated sheet structures that create two domains for each lobe (domains I and II) [1].
Each lobe can bind a metal atom in synergy with the carbonate
ion (CO3 2− ). The metals that it binds are the Fe2+ or Fe3+ ions, but
it has also been observed bound to Cu2+ , Zn2+ and Mn2+ ions [3].
0924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
doi:10.1016/j.ijantimicag.2008.07.020
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S.A. González-Chávez et al. / International Journal of Antimicrobial Agents 33 (2009) 301.e1–301.e8
Table 1
Bacteria against which lactoferrin (LF) has a reported effect
Bacteria
Fig. 1. Three-dimensional structure of biferric bovine lactoferrin at a resolution of
2.8 Angstroms [9].
Because of its ability to reversibly bind Fe3+ , LF can exist free of Fe3+
(apo-LF) or associated with it (holo-LF) [10], and it has a different
three-dimensional conformation depending on whether it is binding Fe3+ [1]. Apo-LF has an open conformation, whilst holo-LF is a
closed molecule with greater resistance to proteolysis [4]. Amino
acids directly involved at the iron-binding site in each lobe are Asp,
Tyr and His, whilst Arg is involved in the bond with the CO3 2− ion
[1]. LF is a basic, positively charged protein with an isoelectric point
of 8.0–8.5 [3].
The primary structure of LF shows the number and position of
Cys residues that allow the formation of intramolecular disulphide
bridges; Asn residues in the N- and C-terminal lobes provide several
potential N-glycosylation sites [1].
3. Biological functions of lactoferrin
Several functions have been attributed to LF. It is considered a
key component in the host’s first line of defence, as it has the ability
to respond to a variety of physiological and environmental changes
[6]. The structural characteristics of LF provide functionality in addition to the Fe3+ homeostasis function common to all transferrins:
strong antimicrobial activity against a broad spectrum of bacteria, fungi, yeasts, viruses [5] and parasites [11]; anti-inflammatory
and anticarcinogenic activities [6]; and several enzymatic functions
[12].
LF plays a key role in maintaining cellular iron levels in the
body, which has been demonstrated with several studies, mostly
in milk. Several decades ago it was shown that breastfed infants
have no iron deficiencies, whilst those fed with ironless paediatric
formulas show a high risk of iron deficiency and related diseases
later in life [13,14]. Also supporting the involvement of LF in this
function is the discovery of LF receptors in the enterocytes of
various species [15] and the high affinity of these receptors for
protein. However, conflicting results have been presented, showing that the lack of these receptors does not affect intestinal
iron absorption [16], which leaves LF’s role in this mechanism
uncertain.
Study
model
Agent
administered
Reference
Gram-positives
Bacillus stearothermophilus
Bacillus subtilis
Clostridium sp.
Haemophilus influenzae
Gram-Negative
Listeria monocytogenes
Micrococcus sp.
Staphylococcus aureus
Streptococcus mutans
In vitro
In vitro
In vitro
In vivo
In vitro
In vivo
In vitro
In vivo
In vitro
hLF
hLF
hLF and bLF
bLF
hLF
hLF
hLF and bLF
bLF
hLF
[5]
[5]
[5]
[17]
[5]
[18]
[5]
[19]
[20]
Gram-negatives
Chlamydophila psittaci
Enteropathogenic Escherichia coli (EPEC)
Enteroaggregative E. coli (EAEC)
Diffusely adherent E. coli (DAEC)
Helicobacter felis
Helicobacter pylori
Legionella pneumophila
Pseudomonas aeruginosa
Shigella spp.
Vibrio cholerae
In vitro
In vitro
In vitro
In vitro
In vivo
In vivo
In vitro
In vivo
In vitro
In vitro
hLF and bLF
hLF
hLF
hLF
rhLF
bLF
bLFa
hLF
hLF
hLF
[21]
[22]
[23]
[23]
[24]
[25]
[26]
[27]
[28]
[5]
Acid–alcohol-resistant bacilli
Mycobacterium tuberculosis
In vitro
hLF
[29]
hLF, human LF; bLF, bovine LF; rhLF, recombinant human LF.
a
For this study, LF was not effective while binding Fe2+ ; it only inhibited the
pathogen when binding Zn2+ or Mn2+ .
3.1. Antibacterial activity
The antibacterial activity of LF has been widely documented
both in vitro and in vivo for Gram-positive and Gram-negative bacteria and in some acid–alcohol-resistant bacteria. Table 1 shows
the bacteria against which LF has shown an inhibitory effect and
the type of LF used. Some of the bacteria listed in Table 1 are specially categorised as antimicrobial-resistant, such as the strains
of Staphylococcus aureus, Listeria monocytogenes and meticillinresistant Klebsiella pneumoniae. LF has also been shown to be
effective against strains of Haemophilus influenzae and Streptococcus
mutans, which can attach themselves to the host cell.
LF’s bacteriostatic function is due to its ability to take up the
Fe3+ ion, limiting use of this nutrient by bacteria at the infection
site and inhibiting the growth of these microorganisms as well
as the expression of their virulence factors [30]. LF’s bactericidal
function has been attributed to its direct interaction with bacterial surfaces (Fig. 2). In 1988 it was shown that LF damages the
external membrane of Gram-negative bacteria through an interaction with lipopolysaccharide (LPS) [31]. The positively charged
N-terminus of LF prevents the interaction between LPS and the bacterial cations (Ca2+ and Mg2+ ), causing a release of LPS from the
cell wall, an increase in the membrane’s permeability and ensuing damage to the bacteria [32]. The interaction of LF and LPS also
potentiates the action of natural antibacterials such as lysozyme,
which is secreted from the mucosa at elevated concentrations along
with LF [33].
LF’s mechanism of action against Gram-positive bacteria is
based on binding due to its net positive charge to anionic molecules
on the bacterial surface, such as lipoteichoic acid, resulting in a
reduction of negative charge on the cell wall and thus favouring
contact between lysozyme and the underlying peptidoglycan over
which it exerts an enzymatic effect [34].
In vitro and in vivo studies have shown that LF has the ability to prevent the attachment of certain bacteria to the host cell.
Attachment-inhibiting mechanisms are unknown, but it has been
S.A. González-Chávez et al. / International Journal of Antimicrobial Agents 33 (2009) 301.e1–301.e8
301.e3
Fig. 2. Mechanism of antibacterial action of lactoferrin (LF). (A) Gram-positive bacteria: LF is bound to negatively charged molecules of the cell membrane such as lipoteichoic acid, neutralising wall charge and allowing the action of other antibacterial compounds such as lysozyme. (B) Gram-negative bacteria: LF can bind to lipid A of
lipopolysaccharide, causing liberation of this lipid with consequent damage to the cell membrane.
suggested that LF’s oligomannoside glycans bind bacterial adhesins,
preventing their interaction with host cell receptors [10].
3.2. Antiviral activity
LF possesses antiviral activity against a broad range of RNA and
DNA viruses that infect humans and animals [3].
Human respiratory syncytial virus is inhibited by LF at concentrations ten times lower than those found in human milk. LF
also acts against non-enveloped viruses such as adenoviruses and
enteroviruses [35].
Human immunodeficiency virus (HIV) remains a major medical
challenge, since current treatment of the syndrome that it causes is
not completely effective. In vitro studies show that, among human
plasma and milk proteins, LF exerts a strong activity against HIV.
This effect is due to inhibition of viral replication in the host cell
[36].
The antiviral mechanisms of LF have not yet been characterised.
LF can block the internalisation of certain viruses into the host cell,
such as poliovirus type 1 which causes poliomyelitis in humans
[37], herpes simplex virus types I and II [38] and cytomegalovirus
[39]. For other viruses, such as hepatitis C virus (HCV) [40] and
rotavirus [41], rather than preventing entry LF inhibits viral replication in the host cell.
Several mechanisms of action have been proposed for LF’s
antiviral effects (Fig. 3). One of the most widely accepted hypotheses is that LF binds to and blocks glycosaminoglycan viral receptors,
especially heparan sulfate (HS). The binding of LF and HS prevents
the first contact between virus and host cell and therefore prevents
the infection [3].
The antiviral effect of LF has also been observed in viruses that
infect animals, such as the Friend virus complex, which causes erythroleukaemia in rodents [42], the feline calicivirus [43] and feline
immunodeficiency virus [44].
3.3. Antifungal activity
LF also has antifungal activity. In 1971, Kirkpatrick et al. conducted the first studies with Candida spp. and attributed the
antifungal effect of LF to its ability to sequester Fe3+ [45]. Later it
was observed that LF can kill both Candida albicans and Candida
krusei by altering the permeability of the cell surface, as it does
with bacteria [46]. Further studies have confirmed LF’s antifungal
activities. In 2003 it was shown that oral LF treatment of oral can-
Fig. 3. Mechanism of antiviral action of lactoferrin (LF). LF can be linked to the viral particle and to glycosaminoglycans, specific viral receptors or heparan sulfate to prevent
internalisation of the virus into the host cell.
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didiasis caused by C. albicans reduces the level of the pathogen and
promotes a cure [47].
Although the antifungal mechanism of action of LF is through a
direct interaction with the pathogen, Fe3+ sequestration is another
important mechanism. In 2007, Zarember et al. showed that Fe3+
sequestration by neutrophil apo-LF is important for host defence
against Aspergillus fumigatus [48].
LF shows an interesting antifungal effect on body tineas caused
by Trichophyton mentagrophytes, against which it acts from a distance. Treatment of guinea pigs with bovine LF (bLF) reduces fungal
infection on the skin of the back and limbs in tinea corpus and tinea
pedis, respectively [49].
3.4. Antiparasitic activity
Most of the studies on LF’s antiparasitic activity have been performed in vitro, assaying molecular associations in the presence or
absence of Fe3+ . This activity has also been shown using peptides
derived from the full molecule.
Intestinal amoebiasis is caused by a protozoan infection and is
one of the leading causes of diarrhoea in children under 5 years
of age and the fourth leading cause of death in the world. The
infection is caused by Entamoeba histolytica, which uses complex
mechanisms to invade the intestinal mucosa and cause amoebic
colitis [50]. Apo-LF is the milk protein with the greatest amoebicidal effect against E. histolytica in vitro, as it can bind the lipids
on the trophozoite’s membrane causing membrane disruption and
damage to the parasite [51].
Other in vitro studies show that hLF can bind the intracellular
parasite Toxoplasma gondii, which causes toxoplasmosis and affects
both humans and animals. However, LF cannot prevent the parasite from entering the host. Its mechanism of action in this case is
inhibition of intracellular growth of T. gondii within host cells [52].
In the case of the haemoparasites Babesia caballi and Babesia
equi, LF’s effect depends on whether or not it is bound to Fe3+ [53].
Babesia caballi was found to be significantly suppressed by apo-LF
but was not inhibited by the other types of LF; for B. equi none of
the LF types showed an inhibitory effect [54].
LF is secreted to the medium, where it shows anti-inflammatory
activity [4] through the inhibition of pro-inflammatory cytokines
such as interferon-gamma, tumour necrosis factor-alpha and interleukin (IL)-1␤, IL-2 and IL-6 [61]. At a cellular level, LF increases the
number of natural killer (NK) cells [62], boosts the recruitment of
polymorphonuclear cells in the blood [63], induces phagocytosis
[64] and can modulate the myelopoietic process [65].
3.6. Anticarcinogenic activity
As in inflammation, LF has the ability to modulate the production of cytokines in cancer. LF can induce apoptosis and arrest
tumour growth in vitro. It can also block the transition from G1
to S in the cell cycle of malignant cells [4].
Treating tumours in mice with recombinant hLF (rhLF) inhibits
their growth by 60% compared with a placebo and increases the
levels of anticarcinogenic cytokines such as IL-18, in addition to
activating NK cells and CD8+ T-lymphocytes [66].
LF’s anticancer effect was recently observed through the immunoexpression of LF in human kidney cell carcinomas and in adjacent
healthy tissue [67]. In vivo studies show that oral administration of
LF results in the inhibition of a T-cell-dependent tumour in head
and neck squamous cell carcinoma [68].
3.7. Enzymatic activity
LF has the ability to function as an enzyme in some reactions.
LF is the milk protein with the highest levels of amylase, DNAse,
RNAse and ATPase activities [12,69]. However, these are not the
only enzymatic activities of LF. The basis for LF’s various enzymatic activities is unknown. However, the variety of activities can
be attributed to variations in the nature of the protein: multiple
isoforms; degrees of glycosylation; tertiary structure (holo- or apoLF); and the degree of oligomerisation. For instance, the LF molecule
capable of hydrolysing RNA has an isoform that is incapable of
binding Fe3+ [70].
The discovery of LF’s enzymatic activities has helped to explain
several of its physiological mechanisms, such as protection against
microbial pathogens, where LF might inhibit growth partly through
hydrolysis of viral, bacterial, fungal and parasitic nucleic acids.
3.5. Immunomodulatory and anti-inflammatory activity
4. Bioactive peptides derived from lactoferrin
LF is a modulator of the innate and acquired immune systems
[55]. Its relationship with the immune system is evident from the
fact that people with congenital or acquired LF deficiency have
recurring infections [56]. In 2006, Wakabayashi et al. used the
quantitative reverse transcriptase polymerase chain reaction (RTPCR) method to assay the expression of 20 immune-related genes
(antimicrobial proteins, pattern recognition receptors and lymphocyte movement related proteins) in the small intestine of mice
administered bLF (2.5 g/kg) and observed that LF can both specifically and non-specifically modulate the expression of those genes
[57].
LF’s positive charge allows it to bind to negatively charged
molecules on the surface of various cells of the immune system [58]
and it has been suggested that this association can trigger signalling
pathways that lead to cellular responses such as activation, differentiation and proliferation. LF has been observed transported into
the nucleus, where it can bind DNA [59,60] and activate different
signalling pathways [4].
In addition to inducing systemic immunity, LF can promote skin
immunity and inhibit allergic responses. It induces the immune
system against skin allergens, causing dose-dependent inhibition
of Langerhans cell migration and the accumulation of dendritic cells
in lymph nodes [6].
Since it was first isolated in 1960 [71], LF has been widely studied
for its antimicrobial characteristics. One of the main mechanisms
used by LF is Fe3+ sequestration. However, it is known that LF may
also interact directly with the pathogen [10]. Enzymatic treatment
of bLF with pepsin produced a low-molecular-weight peptide with
antibacterial properties against a large number of Gram-positive
and Gram-negative bacteria, including Escherichia coli, Salmonella
enteritidis, K. pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa
and Streptococcus bovis. Bellamy et al. [72] identified a region of
amino acids at the N-terminus that retains its biological activity
when separated from the full molecule. This molecule, called lactoferricin B (LFc B), shows greater antimicrobial activity than LF. This
region corresponds to residues 17–41 of bLF [72] and it is now
known that it corresponds to residues 12–48 in several species of
mammals with highly homologous sequences [5].
In characterising the various peptides generated by LF hydrolysis, it was found that minimal variations in the amino acid sequence
change the antimicrobial activity of the peptide. For example,
LFampin 268–284 and LFampin 265–284, chemically synthesised
fragments from the N-terminal sequence of bLF, differ in only three
amino acids (265Asp-Leu-267Ile) but exhibit a different strength of
antimicrobial activity [73].
S.A. González-Chávez et al. / International Journal of Antimicrobial Agents 33 (2009) 301.e1–301.e8
When isolated from the native molecule, the N- and C-termini
show physiological activity through mechanisms independent of
iron sequestration [74].
5. Lactoferrin gene regulation
LF has been identified in several tissues both in humans and
animals and it has extensive homology among species. Its mRNA
levels vary by tissue, suggesting tissue- or cell-specific regulation
[75–78].
To date, the LF gene has been found at the chromosome level in
a set of different species (human chromosome 3 [79] and mouse
chromosome 9 [80]) and its size ranges from 23 kb to 35 kb. The
LF gene is organised in 17 exons, 15 of which are identical in cows,
pigs and mice [81]. In 2008, Kang et al. analysed 60 sequences of
LF genes with full coding regions. They found that the length of
the gene varies widely from species to species, from 2055 to 2190
residues, owing to deletions, insertions and mutations in the stop
codon [78].
LF is expressed both constitutively and inducibly. It is constitutively expressed on mucosal surfaces whilst in some tissues it
301.e5
is induced by external agents. In 2002, oestrogen response elements (EREs) were identified in the promoter of this gene in
humans and mice. In these promoters the ERE overlaps the binding
sites of other transcription factors such as COUP-TF, an oestrogenresponsive negative regulator [82]. Other regulatory factors include
the repressor of oestrogen receptor activity, whose absence has
been proven to increase the expression of oestrogen-induced LF by
up to 100-fold [83]. LF can also be induced by compounds other than
oestrogens, such as retinoic acid, which stimulates gene expression
in embryonic cells [84].
Evidence to date suggests the involvement of multiple signalling
pathways in the regulation of LF expression, and further study may
reveal an even greater complexity of regulatory mechanisms.
6. Clinical applications of lactoferrin
On account of LF’s many functions, it has been tested for clinical
use in disease prevention, treatment and diagnosis.
One of the first applications of LF was in infant formula. Several studies showed that infants fed with infant formulas had less
intestinal iron absorption than breastfed infants [13,14]. Most of
Table 2
Expression of lactoferrin (LF) in various organisms
Organism
Bacteria
Escherichia coli
Rhodococcus erythropolis
Yeasts
Saccharomyces cerevisiae
Pichia pastoris
LF origin
Expression system
Levels of expression
Size
Characteristics
Reference
bLfc
Lfc
bLF C lobe
pET32a vector
Fusion of Lfc and anionic protein genes
pTip LCH1.2 vector
10 mg/L
60 mg/L
3.6 mg/mL
Several
80 kDa
38 kDa
Antimicrobial activity
Antimicrobial activity
Antimicrobial activity
[92]
[93]
[93]
hLF
Chelatin promoter
2.0 mg/L
80 kDa
[94]
hLF
cLF
PIC 3.5 K vector
pGAPZalphaC vector
115 mg/L
2 mg/L
80 kDa
80 kDa
pLF
12 mg/L
78 kDa
bLF
Glyceraldehyde-3-phosphate
dehydrogenase promoter
NR
Antimicrobial activity,
ability to bind Fe3+
Not identified
Ability to bind Fe3+ ,
thermal stability
Ability to bind Fe3+
Antimicrobial activity,
ability to bind Fe3+
Ability to bind Fe3+
[98]
Antimicrobial activity,
ability to bind Fe3+
Immunoreactive, ability to
bind Fe3+
[100]
76 kDa
[95]
[96]
[97]
eLF
pPIC 9 K vector
40 mg/L
80 kDa
Fungi
Aspergillus awamori
hLF
Fusion with the glucoamylase gene
2.0 g/L
80 kDa
Aspergillus oryzae
hLF
Alpha amylase promoter of A. oryzae
25 mg/L
78 kDa
hLF
9.5 mg/L
80 kDa
Ability to bind Fe3+
[102]
hLF
pLF
Infection with nucleopolyhedrosis
virus, P8HLfc vector
Infection with baculovirus vBm-hLF
Infection with baculovirus
65 mg/mL
205 ␮g/pupa
78 kDa
80 kDa
Antimicrobial activity
Biological activity
[103]
[104]
Mammals
Goat
hLF
Microinjection
0.756 mg/L milk
78 kDa
[105]
Mice
hLF
hLF
2 g/L milk
13 mg/mL
80 kDa
80 kDa
Rabbit
Bovine
hLF
hLF
Infection with attenuated adenovirus
Fusion with regulatory elements of the
bovine alphaS1 casein gene
Adenovirus infection
Microinjection
2.3 mg/mL milk
1 g/L milk
80 kDa
80 kDa
Ability to bind Fe3+ ,
temperature and
proteolysis stable
NR
Antibacterial and
anti-inflammatory activity
NR
Antimicrobial activity,
ability to bind Fe3+
Potexvirus infection
pLACMODC/18.1
Agroinfection with Agrobacterium
tumefaciens plasmid: pIG200 and
pIG211
Infection with A. tumefaciens
pUC 18 vector
0.6 % SP
NR
1.6 mg/g seeds
40 kDa
Mixed
78 kDa
Antibacterial activity
Antibacterial activity
Antibacterial activity
[110]
[111]
[112]
0.1% SP
NR
80 kDa
85 kDa
Antimicrobial activity
Adequate glycosylation
level
[113]
[114]
Insects
Spodoptera frugiperda
Bombyx mori
Plants
Nicotiana benthamiana
Nicotiana tabacum
Rice
Potato
Maize
hLF N lobe
hLF
hLF
hLF
hLF
b, bovine; h, human; c, caprine; p, porcine; e, equine; Lfc, lactoferricin; NR, not reported; SP, soluble protein.
[99]
[101]
[106]
[107]
[108]
[109]
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S.A. González-Chávez et al. / International Journal of Antimicrobial Agents 33 (2009) 301.e1–301.e8
the LF is absorbed intact by the infant’s bowel, and thus LF is
distributed by the bloodstream. LF also promotes the proliferation of lactic acid bacteria in the bowel such as Bifidobacterium
and Lactobacillus, which protect the host from harmful bacteria
[85].
The activity of LF and its bioactive peptides has been documented both in vitro and in vivo against a large variety of pathogens.
Studies at a clinical level have considered LF as a possible treatment or prophylactic for a number of diseases. For example, LF
was tested as a second treatment against Helicobacter pylori in
patients with recurring infection. Patients supplemented with bLF
showed a greater recovery from infection [86]. As with antibacterials, LF has shown synergy with antiviral drugs: ribavirin to treat
HCV [87]; cidofovir against cytomegalovirus [3] and zidovudine (an
AZT analogue) against HIV [36]. It has also been observed that LF
added in subinhibitory concentrations to antifungal agents such as
clotrimazole, ketoconazole, fluconazole and itraconazole reduces
the minimum inhibitory concentrations of these agents against
C. albicans. That is, these combinations synergistically inhibit the
growth of the pathogen, with the most resistant strains being the
most sensitive to this combination. The preventive effect of LF has
also been tested. An example is the use of LF as an adjuvant for
the BCG (Bacille Calmette-Guérin) vaccine, where LF enhances the
delayed hypersensitivity response and limits the pathology caused
by Mycobacterium tuberculosis by increasing class II molecule levels
on the surface of antigen-presenting cells and thereby increasing
IL-12 and IL-10 expression [88].
7. Production of native and recombinant lactoferrin
Because of the functional characteristics of LF, attempts have
been made to produce or purify this protein for use as a food additive or therapeutic. Protein purification strategies are based on the
properties of the molecule and depend on three types of chromatography. Since LF has a net positive charge [3], it is efficiently
absorbed on cation exchange resins and is eluted with saline solutions [89] at >95% purity [90]. LF binds Fe3+ so it can be purified
by metal ion affinity chromatography [89]. Furthermore, since LF
is a glycosylated protein it can also be purified by concanavalin A
affinity chromatography [91].
However, the need for larger amounts of LF has led to the development of strategies to obtain a recombinant form of the protein
and, to date, several LF expression systems have been used (Table 2),
including both prokaryotic and eukaryotic organisms. One of the
first expression systems was built in filaments of the Aspergillus fungus, where both hLF and murine LF have been expressed [100,101].
Later, as shown in Table 2, expression systems were developed in
yeasts and bacteria, where the following proteins have been produced: rhLF [94,95]; caprine LF [96]; bLF [98]; equine LF [99]; and
porcine (pLF) [97], as well as LF peptides, including LFc [92,93],
reaching expression levels of 115 mg of hLF/L of fermentate from
Pichia pastoris grown with a high-density batch fermenter [95].
Biotechnological tools have made it possible to use viral vectors
for the expression of LF, mostly through insect infection either in
cell culture or directly in the organism, where the expression of both
hLF and pLF has been successful, resulting in transgenic Bombyx
mori insects expressing 205 ␮g of pLF per infected pupa [103].
LF has also been expressed in higher eukaryotic organisms, both
in plants and animals. Using microinjection and direct infection
with viral vectors in the mammary gland, transgenic animals have
been created that produce milk with rLF. These animals include
goats [105], mice [107], rabbits [108] and cows [109], with expression levels of up to 2 g of hLF/L of milk in transgenic goats. Table 2
also shows the expression systems in plants where hLF expression
has been attained; in rice, 1.6 mg of protein per gram of seed was
produced.
Most of the expression systems that have been developed produce LF or LF peptides in a recombinant form with physical,
biochemical and biological characteristics similar and often indistinguishable from those of native LF, including molecular weight,
degree of glycosylation, antimicrobial and anti-inflammatory activity, thermostability and ability to bind Fe3+ .
These strategies are useful for producing rLF at high levels of
expression for efficient economical large-scale production.
8. Concluding remarks
A wide spectrum of functions have been described for LF. The
beneficial effect of LF in the treatment of various infectious diseases caused by bacteria, fungi, protozoa and viruses in animals
and humans is described above. Despite extensive literature available on LF, the molecular interactions of this protein with regulatory
elements and other proteins for antimicrobial function require further investigation. The great utility of this functional protein has
motivated scientists to overexpress and purify LF from cells as a
potential defence against pathogens.
Funding: This work was supported in part by an internal grant
from Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, México. SAG-C thanks CONACYT for the MC studies grant.
Competing interests: None declared.
Ethical approval: Not required.
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