ABBS 2005,38(03): Recombinant Functional Human Lactoferrin Expressed in Baculovirus System

Abstract        Human lactoferrin (hLf)
is a multifunctional iron-binding glycoprotein. In this study, we amplified hLf
cDNA by reverse transcription-polymerase chain reaction from normal human
mammary gland. The nucleotide sequence of the hLf was identical to the
known hLf. We constructed a recombinant virus, vBm-hLf, harboring the hLf
gene and exploited the BmN cells as host to produce recombinant human
lactoferrin (rhLf). It was found that a recombinant protein with a molecular
mass of approximately 78 kDa was expressed. Approximately 13.5 mg rhLf was purified from 1–2´105 BmN cells infected by vBm-hLf and the rhLf
proved to be biologically active. This method established in our study will
pave the way for efficient production of rhLf for further application of this
protein in the future.

Key words        bacteriostatic activity; baculovirus; Bombyx
mori; human lactoferrin; reverse transcription-polymerase chain reaction

Human lactoferrin (hLf),
an approximately 80 kDa iron-binding glycoprotein, was originally found in
milk. The full-length cDNA of the hLf gene is 2.35 kb, containing an
open reading frame (ORF) of 2136 bp [1]. The protein is widely present in
physiological fluids, such as milk, saliva, tears and bile [2]. It consists of
a single polypeptide chain folded into two structurally homologous lobes that
are linked by a short hinge-like peptide and each lobe has one iron-binding
site [3].

Many functions have been
proposed for lactoferrin, including protection against microbial infection,
regulation of intestinal iron absorption, inhibition of solid tumor growth,
inhibition of hepatitis C virus infection, regulation of translation, and
regulation of immune function [4–9].

Obtaining natural hLf is
rather expensive and difficult because it is present in low concentrations.
Many studies have focused on the heterologous expression of hLf [10–13]. hLf has been expressed in the mammalian
expression system, but the expression level was unstable and not suitable for
fast expression of the proteins. The baculovirus expression system is an inexpensive
high-level expression system. This system has been used effectively for
expression of recombinant proteins, such as hepatitis E virus surface antigen,
human a-interferon,
human growth hormone, human interleukin-2 [14], and many others. It can achieve
high production of heterologous proteins with appropriate posttranslational
modification. As a result, the expressed proteins were correctly folded and
glycosylated and retained their biological activities [15]. Therefore, it
seemed desirable to utilize the baculovirus expression system for the efficient
and economical production of hLf.

In this study, we cloned
the hLf gene from human mammary gland and, for the first time, expressed
functionally active recombinant hLf (rhLf) in baculovirus-infected Bombyx
mori-derived cell line BmN cells.

Materials and Methods

Cell lines and strains

The B. mori-derived
cell line, BmN (gift from Prof. Xiang-Fu WU and conserved by our laboratory),
was cultured at 27 ºC with TC-100 medium (Gibco,  

Reverse
transcription-polymerase chain reaction (RT-PCR)

Total RNA was prepared
and extracted from mammary gland (kindly provided by  

Construction of
recombinant baculovirus

The pBacPAK-hLf was
co-transfected with linearized DNA of B. mori nucleopolyhedrovirus
(BmNPV) BacPAK6 (digested by Bsu36I) into BmN cells (derived
from B. mori) (1´106 cells) to obtain recombinant virus using
Lipofection reagent (Invitrogen) [16]. The recombinant virus was named vBm-hLf,
which was subsequently purified three times by plaque screening and identified
by Southern blotting using Digoxigenin DNA labeling and detection kit (Roche
Applied Science,  

Production and
purification of rhLf in BmN cells

Stock cultures of BmN
cells were grown at 27 ºC with TC-100 medium supplemented with 10% (V/V)
fetal calf serum. Confluent
monolayers were subcultured by removing cells from flasks by gentle pipetting
and dilution (1:3) in complete media. Under production conditions, BmN cells
were cultivated in rhLf was purified from
the cell culture medium by immunoprecipitation using the Seize primary
immunoprecipitation kit (Pierce,  

Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis

The procedures of
electrophoresis, transfer and detection of immunoreactivity were performed as
described previously [17].

Gel electrophoreses were
performed on 12% SDS-PAGE with denaturated samples under reducing conditions.
Protein bands were visualized by Coomassie blue staining. Proteins were
electronically transferred overnight to a polyvinylidene difluoride membrane
(Sigma-Aldrich,  

Enzyme-linked
immunosorbent assay (ELISA) detection of rhLf expression

The ELISA was performed essentially
according to the protocol of Ausubel et al. [18]. The ELISA standard
curve was generated using a series of hLf standard (Sigma-Aldrich) from 1.6
ng/ml to 100 ng/ml according to the manual of Wang et al. [19].

Appropriate 96-well
polystyrene plates ( 

Deglycosylation of rhLf

N-glycosidase F (Roche
Diagnostics Corporation,  

Absorption spectrum

The absorption spectrum
of the purified rhLf was determined by the A280/A465 ratio according to the protocol described by
Parry and Brown [20].

Bacteriostatic activity
of rhLf

To evaluate the
biological activity of rhLf expressed in BmN cells, a time-dependent study was
performed. The non-enteropathogenic E. coli cells were grown to stationary
phase, collected by centrifugation and resuspended in nutrient broth medium.
Then 2 ml bacterial suspension was added to 28 ml of nutrient broth medium to
give a final concentration of 105–106 CFU/ml, and subsequently the rhLf was added
to the medium at 300 mg/ml to
assess its effects on bacterium growth. The mixtures were then incubated on a
shaker (220 rpm) at 37 ºC. The growth of bacteria was evaluated by measuring
the absorbance at 600 nm every 2 h using a spectrophotometer. hLf standard was used
as a positive control and phosphate-buffered saline (PBS) as a negative
control. All experiments were repeated three times.

Statistical analysis

All data are presented
as mean盨D. In all statistical analyses by t-test,
an associated probability of P<0.05 was accepted as the level of statistically significant difference.

Results

Nucleotide sequence
analysis of rhLf

We successfully cloned
the hLf cDNA by RT-PCR estimated to be approximately 2.1 kb. Its nucleotide
sequence showed 99% sequence similarity with that determined by Conneely et
al. (GenBank accession No. AY875691). A Blast (http://www.ncbi.nlm.nih.gov/) search
revealed that the putative rhLf with 711 amino acids had 99% homology with
known hLf. It could be deduced that the rhLf was obtained as an intact protein
and had similar properties to the native hLf [12].

Construction of vBm-hLf
and rhLf expression

We successfully
constructed the recombinant virus, vBm-hLf, using the transfer vector pBacPAK8 which carries a powerful polyhedrin promoter.
In order to confirm the expression of rhLf, BmN cells were infected with BacPAK6 (control) or vBm-hLf. At 120 h
post-infection, the cell culture medium was collected by low speed
centrifugation (According to the hLf standard
curve, the level of rhLf expressed in BmN cells was determined. The result
indicated that the production of recombinant protein was maximal at 120 h
post-infection (approximately 13.5 mg per 1–2´105 cells), after that, the production decreased
rapidly (Fig. 2).

Purification of rhLf and
glycosylation analysis

The culture supernatant
of BmN cells infected with vBm-hLf was collected 120 h post-infection for
purification. The recombinant protein was purified through a series of steps
including reverse-phase column chromatography, HiTrap protein A HP column
chromatography and immunoprecipitation.

The isolated rhLf was
characterized as a single approximately 78 kDa band by Coomassie blue staining.
The molecular mass was approximately 4 kDa lower than that of the hLf standard
(82 kDa). The electrophoretic mobility of the rhLf was slightly faster than
that of the hLf standard. Both the standard and the rhLf showed the same
immunological reactivity with anti-hLf polyclonal antibody.

hLf isolated from milk
contains two N-acetyllactosaminic-type glycans attached through N-glycosidic
linkage. To determine the extent of glycosylation, the recombinant molecule was
treated with N-glycosidase F and resolved by 12% SDS-PAGE. The N-glycosidase F
treatment of rhLf induced an approximately 2 kDa molecular mass decrease. This
value was very close to the calculated molecular mass of the polypeptide chain
(76 kDa). The molecular mass corresponding to that of glycan was evaluated to
be approximately 2 kDa (Fig. 3).

Absorption spectrum of
rhLf

The absorption spectrum
of the purified rhLf was determined. The A280/A465 ratio of the recombinant protein was 23.1,
which indicated that the protein was bound to iron.

Bacteriostatic activity
and its possible mechanism

To evaluate the
bacteriostatic activity of rhLf obtained from the BmN cell culture medium,
time-dependent studies were conducted. In this experiment, purified rhLf was
added into E. coli K-12 cell culture medium. the hLf standard was
used as a positive control and PBS as a negative control. The results from Fig.
4 show that rhLf was slightly more inhibitory than the hLf standard under
the same assay conditions. No significant difference (P>0.05) between
the standard hLf and rhLf for the E. coli strain was found. The
difference between the rhLf and the negative control was significant (P<0.05), indicating the successful expression of rhLf in BmN cells with high bioactivity.

To understand the
possible mechanism of rhLf in bacteriostatic activity, the bacterial cells
treated with rhLf were observed. The dead cells showed irregular shape and part
of the cell membrane was found to be damaged. The structure of the bacterial
cells changed from a regular rod-shaped form to a compressed one. This
indicated the leakage of cell contents, which could be attributed to
nutritional deficiency, particularly a lack of iron, which shows the direct
interaction of lactoferrin with bacteria.

Discussion

The rhLf has been expressed
in transgenic cows, mammalian BHK cells, Spodoptera frugiperda (Sf9)
cells and tobacco plants, to name a few [10–13]. However, the BHK system is time consuming, requires clonal
selection and sophisticated culture conditions, and thus is not suitable for
fast and easy production of recombinant proteins. Other expression systems,
such as yeast and bacteria, as described previously are not stable [12]. The
baculovirus expression system has been proven to be a most effective and
versatile eukaryotic expression tool [21]. Since the first report by Maeda et
al. on the production of human a-interferon
in B. mori silkworm larvae [22], it has been used to express many
recombinant proteins using insects as bioreactors, such as hepatitis E virus
surface antigen, human growth hormone and human interleukin-2 [14].

We successfully cloned
the hLf cDNA by RT-PCR. A Blast search revealed that the deduced
sequence of rhLf was similar to that of the native hLf, indicating that the
correct processing of the signal peptide and identical posttranscriptional
processing of the signal sequence occurred. We suggest that the changes in
amino acids are mostly caused by natural genomic polymorphisms.

A high and stable
expression level of functional rhLf in BmN cells (13.5 mg per 1–2´105 cells) was reported, which is in accordance
to that of Salmon et al. [12]. This is the first time its expression in
BmN cells has been reported. The rhLf was released into the cell culture medium
and our attempt to purify the protein using conventional reverse-phase column
chromatography and immunoprecipitation was successful. The production level of
the recombinant protein was maximal at around 120 h post-infection and then
decreased rapidly. The degradation of the rhLf might be the result of over
infection of recombinant viruses, which resulted in the release of endogenous
proteases. Another possible explanation was that a cysteine protease encoded by
the BmNPV causes degradation of the recombinant proteins during the later
stages of the infection [23].

Estimation of the
molecular mass of rhLf was achieved by SDS-PAGE and Western blotting, and we
found that the isolated rhLf was visualized as a single, approximately 78 kDa,
Coomassie blue stained band. N-glycosidase F treatment of rhLf showed a
decrease of 2 kDa in molecular mass, which may be because the rhLf expressed in
BmN cells have incomplete glycan structures compared with those expressed in
mammalian cells. The discrepancy in the apparent molecular mass was therefore
most likely due to the difference in the glycosylation pattern or to the
partial degradation of the protein by the host proteases secreted in response
to the recombinant virus.

In order to clarify if
rhLf expressed in BmN cells was biologically active, we also studied the
bacteriostatic effects of rhLf. The results indicated that rhLf and hLf
standard were bacteriostatic toward E. coli and the antibacterial
activity of rhLf was slightly stronger than that of the hLf standard. This was
most likely attributable to the distinct glycosylation, which was essential to
the protein functions. Accordingly, rhLf is expected to be used as a natural
antibacterial material in general and clinical foods, drugs and cosmetics.
Further studies concerning the mechanism of this activity are now in progress.

For decades, the
silkworm and pupa were used as ideal bioreactors to produce foreign proteins.
We have expressed the rhLf in silkworm successfully [24]. Compared with BmN cells
and silkworm, the pupas are more easily manageable and convenient for gene
operation. Also, the protein expression level in pupa is 10- to 1000-fold
higher than that in BmN cells. The BmNPV expression systems are much safer than
other known systems as they are noninfectious to the animals belonging to other
groups. The protein expression in BmN cells is the first step for its
expression in pupa successfully. We hope that the method established in our
studies will pave the way for efficient industrial production of functionally
active rhLf on a large scale for further utilization of pupa as a bioreactor.

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