ORI GI NAL ARTI CLE

Expression and purication of a recombinant Fip-fve

protein from Flammulina velutipes in baculovirus-infected
insect cells
C.-M. Wu
1
, T.-Y. Wu
2
, S.-S. Kao
1
, J.-L. Ko
3
and T.-R. Jinn
1
1 Biopesticide Department, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Wufeng, Taiwan
2 Department of Bioscience Technology, Chung Yuan Christian University, Chungli, Taiwan
3 Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung, Taiwan
Introduction
Fip-fve is an immunomodulatory protein isolated from
Flammulina velutipes (Ko et al. 1995). The Fip-fve is
classed as a new family of fungal immunomodulatory
proteins (Fips) that contain LZ-8, Fip-vvo and Fip-gts,
which can be isolated and puried from Ganoderma luci-
dum (Lingzhi), Volvariella volvacea and Ganoderma tsu-
gae, respectively (Kino et al. 1989; Hsu et al. 1997; Lin
et al. 1997). Fips exhibit a high homology in their amino
acid sequences (Hsu et al. 1997), which comprise 110
amino acid residues with a molecular mass of 13 kDa,
and are signicantly similar to the V
H
region of the
immunoglobulin in both amino acid sequence and pre-
dicted secondary structure (Tanaka et al. 1989). Fips
are mitogens in vitro for human peripheral blood lym-
phocytes (hPBLs), and are mediated by active macro-
phage and T-lymphocytes to induce the production of
cytokine, interleukin 2 (IL-2), IL-8, interferon-c and
tumour necrosis factor-a (Haak-Frendscho et al. 1993;
Wang et al. 1997). Various therapeutic effects of
Fips, have been noted, they include immune-regulating,
anti-tumour, hepatoprotective, anti-virus, antioxidant,
cholesterol-lowering, hypoglycaemia-induction and
Keywords
allergy, baculovirus insect cell expression
system, Flammulina velutipes, fungal
immunomodulatory protein, interleukin 2,
nickel-afnity chromatography.
Correspondence
Tzyy-Rong Jinn, Biopesticide Department,
Taiwan Agricultural Chemicals and Toxic
Substances Research Institute, 11 Kuang-Ming
Road, Wufeng, Taichung Hsien 41301,
Taiwan. E-mail: jihn@tactri.gov.tw
2007 1136: received 17 July 2007, revised 8
October 2007 and accepted 13 October 2007
doi:10.1111/j.1365-2672.2007.03686.x
Abstract
Aims: To develop an efcient and facile expression system supply of high pur-
ity and stable activity of rFip-fve for oral administration, medicinal study and
applications.
Methods and Results: A recombinant virus that contained the chimera gene,
encoding a bombyxin signal peptide sequence fused to a Fip-fve-6His sequence,
was constructed. The rFip-fve was puried from the supernatant of the infected
Sf21 cells using a nickel-chelated afnity column, and was veried by Western
blot and MALDI-MS (matrix-assisted laser desorption ionization mass spectro-
metry) analyses. Results showed that a glycosylated mature rFip-fve was pro-
duced and secreted into the infected cell supernatant. The immunomodulatory
activity of rFip-fve was evaluated by measuring the amount of interleukin-2
released from murine splenocytes.
Conclusions: A reliable scheme to express and purify active rFip-fve in a bacu-
lovirus insect cell system for medicinal applications and genetic study is a fea-
sible means of solving potential problems related to the production and
activity of rFip-fve protein.
Signicance and Impact of the Study: The rFip-fve expressed in insect cells
was processed and modied in a manner more similar to that of its native
counterpart than that in bacterial cells. Therefore, the potential applications of
rFip-fve that is generated in Sf21 cells can be more effectively evaluated that
produced in Escherichia coli.
Journal of Applied Microbiology ISSN 1364-5072
1354 Journal compilation 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 13541362
2008 The Authors
appetite-suppression effects (Wasser and Weis 1999;
Mau et al. 2002).
Of these Fips, the native Fip-fve comprises 114 amino
acid residues, has a molecular mass of 12 704 Da, and
with LZ-8 has a high degree of amino acid sequence
homology, with 70 (614%) invariant amino acid residues
(Ko et al. 1995). The native Fip-fve is a glycoprotein, in
which a potential N-glycosylation site is present at posi-
tion 54 (Ko et al. 1997). The immunomodulatory activity
of Fip-fve was examined by investigating its effects on
systemic anaphylaxis (Hsieh et al. 2007). The Fip-fve
could induce the Th1-skewing in the development of the
allergen-specic immune response, and efciently sup-
pressed systemic anaphylaxis (Hsieh et al. 2007). Fip-fve
may become a new therapeutic agent for allergic diseases.
However, little information is available on the expression
and purication of the Fip-fve protein for medicinal
applications.
As reported elsewhere, the baculovirus insect cell
expression system is now widely employed to produce
recombinant glycoproteins (Congote and Li 1994; Hooker
et al. 1999). The N-glycan processing pathways are pres-
ent in insect cells (Jarvis 2003; Kato et al. 2004; Kost et al.
2005). Additionally, the baculovirus has a restricted host
range, is noninfectious to vertebrates and does not gener-
ate pathogenic or toxic compounds in humans (Groner
1986). So, the baculovirus insect cell system is an ideal
system for expressing foreign proteins for therapeutic or
medicinal applications.
In this study, the rFip-fve was processed, and then
secreted in culture medium to facilitate the purication
of rFip-fve proteins. This study also is the rst in which
Fip-fve was successfully expressed in a system of baculovi-
rus insect cells.
Materials and methods
Insect cell line
IPLB-Sf21-AE (Sf21; Invitrogen, Carlsbad, CA), a cell line
originally isolated from the ovaries of the fall armyworm,
Spodoptera frugiperda, was employed herein. Sf21 cells
were maintained at 26C in TNM-FH basal medium
(Sigma) that was supplemented with 10% heat-inactivated
foetal bovine serum (FBS) (Summers and Smith 1987).
Generation of recombinant baculovirus
The pGEX-Fve, which contained the entire coding
sequence of Fve (Met1Thr115), was kindly provided by
Dr Ko (from the Institute of Medical and Molecular
Toxicology, Chung-shan Medical University, Taiwan).
First, the Fve (Ser2Thr114) fused with an additional
6His tag at the carboxyl terminus was obtained using a
PCR with primers 5-CGAGATCTTCCGCCACGTCGC-
TC-3 (BglII site underlined) and 5-ACGAATTCTAA-
TGGTGATGGTGATGGTGAGTCTTCTTCCACTCAGC-3
(EcoRI site underlined). The resulting PCR fragment was
introduced at the BglII and EcoRI restriction sites of the
transfer vector pAcUW21 (PharMingen, San Diego, CA),
and thus directed by the p10 promoter. The resulting
plasmid was designated pAcP
10
Fve (without signal pep-
tide) (data not shown). Consequently, an 81 bp nucleo-
tide sequence, encoding the 19 amino acid bombyxin
signal peptide (SP
bbx
) signal peptide and the N-terminal
four amino acids from the neuropeptide bombyxin (Gen-
Bank accession no. D00340) (Kondo et al. 1996), was
synthesized by the Bio-Basic company (Ontario, Canada),
and contained BglII BglII restriction sites. Following
treatment with endonucleases BglII, the generated BglII
SP
bbx
DNA fragments were ligated to the BglII-digested
pAcP
10
Fve, generating the recombinant plasmid
pAcP
10
SP
bbx
Fve which is the SP
bbx
sequence in frame
fused with: Fve6His (Fig. 1). The recombinant plasmid
was identied by restriction endonuclease analysis and
DNA sequencing. A recombinant baculovirus,
vAcP
10
SP
bbx
Fve, was generated from Sf21 cells that were
co-transfected with 1 lg plasmid DNA and 02 lg linear-
ized AcRP23.LacZ DNA (PharMingen) using lipofectin.
They were then puried by three rounds of plaque assay
to harvest single and stable recombinant baculovirus. Pla-
que titration of the virus was performed according to the
standard protocol described by OReilly et al. (1992).
Expression of rFip-fve in baculovirus insect cell system
Sf21 cells were initially seeded into a cell culture ask
(2 10
6
25 cm
2
ask) with approx. 80% conuence.
After the cells had attached, the medium was removed
and the cells were infected with vAcP
10
SP
bbx
Fve at multi-
plicities of infection (MOI) of 10 for 48 h at 26C. To
test optimal condition for production of rFip-fve in bacu-
lovirus insect cell expression system, Sf21 cells were
seeded into six well plates (1 10
6
cells per well) with
approx. 85% conuence to optimize the production of
rFip-fve. The cells were infected with vAcP
10
SP
bbx
Fve at
various MOI (1, 5 and 10). Infection was carried out at
26C for 48, 72 and 96 h. At the end of infection, the cul-
ture supernatants were harvested and then concentrated
to a nal volume of 200 ll using a 10 kDa cut-off con-
centrator (Millipore, Billerica, USA). The identity of the
samples was analysed by SDS-PAGE, glycoprotein staining
and Western blot using anti-His serum. The expressed
rFip-fve from supernatant was quantitatively analysed by
scanning and digitizing the immunoblotting membrane
using an Alphaimager image-analyzing system (Alpha
C.-M. Wu et al. Functional expression of Fip-fve
2008 The Authors
Journal compilation 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 13541362 1355
Innotech, San Leandro, CA). One microgram of puried
rFip-fve protein was used as a reference for calculation.
Data were collected from triplicate experiments, and the
resulting values were averaged and analysed using one-
way anova using jmp 5.01.
SDS-PAGE, glycoprotein stain and Western blot analyses
Samples were prepared for SDS-PAGE by mixing 15 ll
aliquots with an equal volume of sample buffer. All sam-
ples were boiled for 5 min and stored at 4C before elec-
trophoresis. SDS-PAGE was conducted in 15% PAGE and
visualized by staining with Coomassie brilliant blue G250.
A glycoprotein staining assay was conducted in SDS-PAGE
as described by the manufacturers of the Gelcode

glyco-
protein staining kit (Pierce). For Western blot analysis,
proteins that were resolved in SDS-PAGE were transferred
to a PVDF (polyvinylidene uoride) membrane (Perkin-
Elmer, Wellesley, USA) in a Bio-Rad Trans-Blot system,
following the manufacturers instructions. The membrane
was subjected to immunodetection using anti-His IgG
antibody (Rockland, Gilbertsville, PA) and alkaline phos-
phatase-conjugated goat anti-rabbit IgG antibody (Sigma)
as primary and secondary antibodies, respectively.
Purication of rFip-fve
6His-tagged rFip-fve protein from the culture supernatant
of vAcP
10
SP
bbx
Fve-infected Sf21 cells was puried as
described by van der Geld et al. (2002) with some modi-
cations. All purication steps were conducted at 4C.
Cell-free culture supernatant that contained rFip-fve was
dialysed against phosphate-buffered saline (PBS). Next,
the dialysed supernatant was combined with 5 ml of 50%
Ni-NTA slurry (Novagen, Darmstadt, Germany) in bind-
ing buffer. The column was then washed with ten vol-
umes of lysis buffer and six volumes of wash buffer (500
NaCl, 20 TrisHCl and 60 mmol l
)1
imidazole, pH 79).
Elution was carried out by adding 5 ml of elute buffer,
supplemented with 250 mmol l
)1
imidazole.
MALDI-MS of rFip-fve
The expected protein band of rFip-fve resolved in SDS-
PAGE was manually excised from the gel and ground into
pieces. The gel pieces were washed twice with 50% aceto-
nitrile and 50% acetonitrile 25 mmol l
)1
ammonium
bicarbonate. The protein in the gel was then reduced
and alkylated at 56C for 45 min in 10 mmol l
)1
dithio-
threitol and 55 mmol l
)1
iodoacetamide in 25 mmol l
)1
ammonium bicarbonate. It then underwent in-gel diges-
tion overnight at 37C with 01 lg of TPCK (N-tosyl-l-
phenylalanine chloro methyl ketone)-treated modied
porcine trypsin (Promega, Madison, WI) in the same buf-
fer. The supernatant, containing resulting tryptic peptide,
was combined with those extracted twice from the gel
pieces by 50% acetonitrile 5% formic acid, and subjected
to MALDI-MS analysis using a quadrupole-time-of-ight
mass spectrometer (Micromass Q-Tof Ultima, Taichung,
Taiwan) at the Biotechnology Center of National Chung-
Hsing University, Taiwan.
Expression and purication of rFip-fve in Escherichia coli
BL21(DE3) cells
Glutathione S-transferase:rFip-fve (GST:rFip-fve) fusion
protein was expressed in E. coli BL21(DE3) cells that car-
ried a recombinant pGEX-Fve plasmid, as described by
Ko et al. (1997). For purication, the expressed GST:rFip-
fve was directly placed on a 2 ml glutathione-Sepharose
4B column, washed with 20 ml of PBS and then eluted
polh gene
f1 ori
Amp
R
ColE1 ori
polh promoter
SPbbx Fip-fve gene
Bgl II
EcoRI
p10 promoter
pAcP10-SP:fve
9719 bp
Figure 1 Schematic diagram of construction of pAcP
10
SP
bbx
fve. Sym-
bols: hatched arrow indicates p10 promoter and Fip-fve gene; solid
arrow indicates SP
bbx
gene; open arrow indicates polhedrin (polh) pro-
moter and polh gene. The lower panel display the DNA sequence of
SP
bbx
-Fve-6His and its deduced protein sequence SP
bbx
(dashed line)
and N-terminal 14 amino acid of bombyxin protein (hatching) and
6His tag (solid line).
Functional expression of Fip-fve C.-M. Wu et al.
1356 Journal compilation 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 13541362
2008 The Authors
with 5 mmol l
)1
reduced glutathione in 10 mmol l
)1
TrisHCl buffer (pH 80). After the GST:rFip-fve was
treated with thrombin protease for 24 h at 22C to release
rFip-fve from the fusion protein.
Measurement of IL-2 in mouse splenocytes
The biological activity of rFip-fve was measured as IL-2
was released from murine splenocytes. Briey, splenocytes
of mice (Balb C; 810 weeks old) were isolated by the
FicollHypaque procedure (Giraudo et al. 1976) and
resuspended to 1 10
7
cells per ml in RMI 1640 medium
(GibcoBRL, Frederick, MD) that had been supplemented
with 10% FBS, 100 units per ml penicillin, 200 mmol l
)1
l-glutamate and 10 mmol l
)1
HEPES, pH 73. To 01 ml
of cells (1 10
6
cell per well) that was seeded on to the
wells of a 96-well plate (Nunc, Rochester, NY), and 2 lg
of rFip-fve in 01 ml of the RPMI 1640 medium was
added to a nal rFip-fve concentration of 10 lg ml
)1
.
Commercial concanavalin A (Con A), a known T-cell
mitogen that induced cytokine production in lymphocytes
(Wermerskirchen et al. 2000), was used as a positive con-
trol at a concentration of 5 lg ml
)1
. Following incubation
at 37C under 5% CO
2
for 48 h, the culture supernatants
were collected. The levels of IL-2 in the supernatants were
measured using a commercial IL-2 cytokine protein array
that contained IL-2 murine cytokines (Biosource, Camar-
illo, CA) with a solid-phase ELISA, as described by the
manufacturers. A serial dilution of recombinant mouse
IL-2 was prepared as a standard to quantify IL-2 cytokine
concentration. Each well in the 96-well plate was coated
with 100 ll of diluted capture antibody (1 lg ml
)1
bioti-
nylated goat anti-mouse IL-2 diluted in PBS) and incu-
bated at room temperature for 2 h. After three washing
steps, each well in the plates was developed with 100 ll
streptavidinhorseradish peroxidase for 20 min, and then
measured immediately at a wavelength of 450 nm using a
microplate reader (TeKon Technologies, Irvine, CA).
Results
Generation of recombinant baculovirus vAcP
10
SP
bbx
Fve
Figure 1 depicts a schematic presentation of recombinant
plasmid pAcP
10
SP
bbx
Fve. A chimera gene that encodes a
SP
bbx
and an N-terminal 14 amino acid of the bomb-
yxin was fused to a Fip-fve-6His sequence. Then this
chimera gene was engineered into the genome of a poly-
hedron-positive AcMNPV under the control of the p10
promoter. The resulting recombinant AcMNPV was
named vAcP
10
SP
bbx
Fve. The recombinant baculovirus
vAcP
10
SP
bbx
Fve was constructed to generate rFip-fve pro-
tein in Sf21 insect cell culture medium. We identied a
high-efcacy clone from six clones by immunoblot with
anti-His antibody and determine the titer of stocks to
be around 69 10
7
plaque-forming units per ml
(PFU ml
)1
) for the production of protein.
Expression of rFip-fve in Sf21 cells
The culture medium that was infected with recombinant
virus vAcP
10
SP
bbx
Fve at an MOI of ten for 24 and 48 h
was collected to evaluate rFip-fve protein expression in
Sf21 cells. The sample proteins were separated on a 15%
SDS-PAGE. An extra protein band 14 kDa, which
matched to the molecular mass of the 6His-tagged rFip-
fve, was detected. This protein was weak at 24 h postin-
fection, and became strong at 48 h postinfection in the
infected culture medium. No protein band was detected
in uninfected Sf21 cell culture medium (Fig. 2, upper
panel). Western blot analysis using anti-6His antibodies
yielded a similar result, exhibiting an immunoreactive
band at around 14 kDa (Fig. 2, lower panel: the mem-
brane seems to have two bands) in the culture medium
of vAcP
10
SP
bbx
Fve-infected Sf21 cells. No immunoreactive
band was detected in uninfected Sf21 culture medium.
Moreover, the putative amount of secreted rFip-fve was
evaluated, regardless of whether it is glycosylated, using a
glycoprotein staining assay. The results revealed that the
putative rFip-fve protein band 14 kDa in the vAcP
10
SP
bbx
Fve-infected supernatant fraction was stained as a
magenta band and no protein band was stained in the
recombinant E. coli BL21 (DE3) cells that carried a
recombinant pGEX-GST:rFip-fve plasmid (Fig. 3, upper
panel). The identity of the samples was also analysed on
SDS-PAGE by Coomassie blue staining (Fig. 3, lower
panel). The results indicated that the addition of SP
bbx
signal peptide enables insect cells to generate a mature
and glycosylated form of rFip-fve. The mature rFIP-fve is
expressed as a glycosylated protein and is secreted into
the culture medium of vAcP
10
SP
bbx
Fve-infected Sf21 cells.
Purication and MALDI-MS identication of rFip-fve
The infected cell supernatant was collected for further
purication using nickel-chelated afnity chromatography
(NTA-Ni
2+
) under native conditions, to purify the puta-
tive rFip-fve expressed in Sf21 cells. The puried product
exhibited a major band with an apparent molecular mass
of 142 kDa by SDS-PAGE. The putative rFip-fve in the
SDS-PAGE gel appeared to be around 94% pure (Fig. 4).
Notably, at 48 h postinfection, the major protein at
142 kDa and a minor band at 14 kDa were immuno-
detected. (The immunodetected protein pattern is similar
to that presented in Fig. 2 (lower panel)). Two putative
rFip-fve bands were detected in these gels. This may be
C.-M. Wu et al. Functional expression of Fip-fve
2008 The Authors
Journal compilation 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 13541362 1357
resulted by different levels of heterologous glycosylation, a
phenomenon commonly occurs in insect cells (Fiebich
et al. 1993; Congote and Li 1994). The purity of the puta-
tive rFip-fve protein band at 142 kDa was further anal-
ysed by MALDI-MS. The results indicated that the four
peptide fragments cumulatively correspond to 65 amino
acid residues of Fip-fve, and over half of the protein
sequence could be veried by the MALDI-MS analysis
(Fig. 4b). These results demonstrated that the puried
142 kDa protein was the mature rFip-fve and this
protein was correctly processed and secreted into the
supernatant of infected Sf21 cells. Table 1 summarizes the
purication of rFip-fve from insect cells. The rFip-fve
recovery yield in one step using the NTA-Ni
2+
afnity
column was nearly 62% and around 625 mg puried
rFip-fve was obtained from 1 l of culture supernatant
equivalent to 31 lg rFip-fve from 1 10
6
infected Sf21
cells.
Optimal conditions for producing rFip-fve in infected
Sf21 cells
Sf21 cells were infected with vAcP
10
SP
bbx
Fve at various
virus titers and culture supernatant harvested at various
kDa 24 48 (h) M
a
r
k
e
r
U
n
i
n
f
e
c
t
e
d
S
f
2
1

c
e
l
l
s
S
f
2
1

c
e
l
l
s
s
u
p
e
r
n
a
t
a
n
t
s
u
p
e
r
n
a
t
a
n
t
I
n
f
e
c
t
e
d
178
120
80
53
40
27
20
15
53
40
27
20
15
9
Figure 2 SDS-PAGE and Western blot analysis of rFip-fve expression
in Sf21 cells supernatant. Proteins (15 lg) from the virus
vAcP
10
SP
bbx
fve-infected Sf21 cells supernatant, and from the nonin-
fected Sf21 cells supernatant were resolved on SDS-PAGE gel by Coo-
massie blue staining (upper panel). A duplicate gel was transferred to
the PVDF membrane and then subjected to immunoblotting using
anti-His antibody (lower panel). A solid arrow indicates the position of
rFip-fve.
Marker
120
86
47
34
26
20
kDa
kDa
120
86
47
34
26
20
1 2 3 4 5
Figure 3 Glycoprotein staining analysis of rFip-fve in Sf21 cells super-
natant. Sf21 cells were infected with virus vAcP10SP
bbx
fve at an MOI
of 10 for 48 h. The infected cells supernatant was resolved on 15%
SDS-PAGE gel, and then was stained using glycoprotein staining kit
(upper panel) and Coomassie blue staining (lower panel). Additionally,
5 lg of horseradish peroxidase and soybean trypsin inhibitor were
used as positive and negative controls, respectively. Lane 1, horserad-
ish peroxidase; lane 2, soybean trypsin inhibitor; lane 3, uninfected
Sf21 cells supernatant; lane 4, infected Sf21 cells supernatant; lane 5,
GST:rFip-fve in Escherichia coli. The solid arrow and the open arrow
indicate the position of the mature form of rFip-fve from insect cells
and GST:rFip-fve fusion protein from E. coli, respectively.
Functional expression of Fip-fve C.-M. Wu et al.
1358 Journal compilation 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 13541362
2008 The Authors
times. The expressed rFip-fve was collected from infected
culture medium, detected on Western blot and evaluated
by the densitometric scanning of the immunologically
recognized band 142 kDa and then was compared with
1 lg puried glycosylated rFip-fve. When the Sf21 cells
were infected with vAcP
10
SP
bbx
Fve at MOI = 1, 5 and 10,
the concentrations of rFip-fve were 254, 426 and
614 mg l
)1
at 48 h, and 56, 1014 and 642 mg l
)1
at
72 h, 421, 662 and 204 mg l
)1
at 96 h (Fig. 5). In this
investigation, the degradation of rFip-fve and the decom-
position of insect cells were also observed at 96 h postin-
fection. Accordingly, the glycosylated rFip-fve reached the
highest level between 72 and 96 h postinfection and the
optimal MOI was 5 (Fig. 5).
Effect of rFip-fve on cytokine release from murine
splenocytes
rFip-fve was expressed and puried from E. coli following
the protocol as reported previously (Ko et al. 1997). To
compare the immunomodulatory activities of rFip-fve
proteins that are expressed in Sf21 insect cells and E. coli
cells. IL-2 cytokine was released from murine splenocytes
as an assay system using Con A, which is a T-cell mitogen
that is known to induce the production cytokine in lym-
phocytes as a positive control (Wermerskirchen et al.
2000).
The results demonstrated that the amounts of IL-2 pro-
duced using equal quantities (2 lg; 10 lg ml
)1
) of rFip-
fve that had been puried from Sf21 insect cells and E.
coli cells were 503 and 1653 pg ml
)1
, respectively
(Fig. 6). Evidently, the rFip-fve produced in Sf21 insect
cells exhibited stronger specic immunomodulatory activ-
ity than those produced in E. coli cells. The positive con-
trol, Con A, of 5 lg ml
)1
clearly enhanced the expression
of IL-2 to a level of 12348 pg ml
)1
, which falls within
the range 971214985 pg ml
)1
, reported previously
(Wermerskirchen et al. 2000). Meanwhile, the detected
levels of IL-2 induction by 5, 10, 15 and 20 lg ml
)1
of
puried rFip-fve from Sf21 insect cells were 349 508,
503 421, 6681 621 and 8036 422 pg ml
)1
,
respectively (data not shown).
kDa
M
a
r
k
e
r
P
u
r
i
f
i
e
d
r
F
i
p
-
f
i
v
e
53
40
27
20
15
9
(a)
(b)
Figure 4 SDS-PAGE and MALDI-MS analysis of puried rFip-fve. rFip-
fve was puried by nickel-chelated afnity chromatography under
native conditions. (a) The purity of the harvested rFip-fve was exam-
ined on 15% SDS-PAGE gel. (b) Afnity-puried rFip-fve was sub-
jected to protein identication by MALDI-MS analysis. Four peptide
fragments of rFip-fve identied by MALDI-MS analysis are underlined.
Table 1 Purication of rFip-fve
Purication
step
Total protein
(mg)*
Total rFip-fve
protein (mg)
Recovery
(%)
Purication
factor
Conditional
media
1140 1015 100 1
Ni-NTA 681 625 616 1031
*Total protein mass was estimated using a protein assay kit (Sigma)
with BSA as a standard.
rFip-fve protein mass was determined by densitometric analysis of
results.
One litre of culture media was used to purify rFip-fve.
Time post-infection (h)
r
F
I
P
-
f
v
e

p
r
o
t
e
i
n

p
r
o
d
u
c
t
i
o
n

(
m
g

l

1
)
48
0
2
4
6
8
10
12
72 96
Figure 5 Time course of optimal production of rFip-fve. The optimal
condition for rFip-fve expression was determined from the culture
supernatant of virus vAcP
10
SP
bbx
fve-infected Sf21 at three MOI values
(, 1; ., 5 and n, 10). These data were obtained from three sepa-
rate experiments and are presented as mean SD.
C.-M. Wu et al. Functional expression of Fip-fve
2008 The Authors
Journal compilation 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 13541362 1359
Discussion
Fip-fve, a Fip, has a potential to modulate an immune
response that is similar to that of LZ-8 (Curtis et al.
2002; Ng et al. 2006) which is a mitogen for hPBLs and
has a bell-shaped doseresponse curve that is similar to
that of lectin mitogen (Haak-Frendscho et al. 1993). Fip-
fve exhibits immunomodulate and immunoprophylactic
activities against allergic diseases (Hsieh et al. 2003,
2007). Since numerous allergy-therapeutic drugs, such as
cyclosporine, prednisolone and FK506, show toxic effects
on pancreatic islets while Fip-fve does not (Hsieh et al.
2003). Fip-fve is a good candidate for developing new
therapeutic agents for treating allergy or autoimmune dis-
eases. Even if the market price of F. velutipes is a rela-
tively cheap, the direct extraction of native Fip-fve from
the F. velutipes mushroom is not feasible for medicinal
applications because the physiological activities of native
Fip-fve depend upon the strain used and the process of
cultivation (Dong and Shi 2006). In addition, the Fip-fve
puried procedure from the F. velutipes mushroom is rel-
atively uneconomic, time-consuming and nally identied
by HPLC on a C18 column (Ko et al. 1997). Additionally,
utilize the project of genetic engineering to elucidating
the functional domain of Fip-fve or developing a mutant
rFip-fve that produces higher activity than native Fip-fve
is also very important issue for oral administration and
medicinal applications in future. Thus, a facile and steady
expression system supply of high-purity and stable-active
Fip-fve is indispensable for production and study of Fip-
fve. As reported elsewhere, puried rFIP-fve has also been
expressed in E. coli, but with an activity that was lower
than that of native Fip-fve. The concentration that maxi-
mizes the stimulatory activity of native Fip-fve is
40 lg ml
)1
, but that for puried rFIP-fve from E. coli is
100 lg ml
)1
(Ko et al. 1997). A similar result was also
observed when the rFip-gts expressed in E. coli was not
folding properly and without glycosylation, retaining its
complete active structure (Jinn et al. 2006). Furthermore,
the rFip-fve extracted from E. coli appeared to be
unsuited for oral administration due to the potential
problem of endotoxin contamination in the protein prep-
aration. So, a reliable scheme to express and purify active
rFip-fve in a baculovirus insect cell system for medicinal
applications and genetic study is a feasible means of solv-
ing potential problems related to the production and
activity of rFip-fve protein. The baculovirus insect cell
system is widely regarded as an excellent tool for produc-
ing recombinant glycoproteins (Jarvis 2003). SP
bbx
has
been demonstrated to be capable of directing various
eukaryotic proteins expression and secreting into culture
medium (Congote and Li 1994; Ma et al. 1998; Rajendra
et al. 2006). Moreover, the recombinant protein is
secreted into the culture medium, substantially allowing
for easy purication (Choo et al. 2002). Accordingly, we
inserted the synthetic SP
bbx
with the N-terminal 14
amino acids of bombyxin into the Fip-fve N-terminus. As
expected, a large amount of processed and glycosylated
mature rFip-fve was produced and secreted into the
infected cell supernatant. The yield of the produced rFip-
fve was 625 mg l
)1
in the infected Sf21 cells supernatant
which was higher than the 5mg l
)1
yield of rFip-fve pro-
duced from E. coli (Ko et al. 1997). In this work, rFip-fve
was expressed under the control of the very late p10 pro-
moter, rather than the polyhedrin promoter, such that
polyhedra remained intact and are expected to elevate the
efciency of primary infection of insect larvae (Trichoplu-
sia ni) in follow-up research, which will be undertaken to
reduce cost of rFip-fve production. The expression of IL-
2 on the lymphocyte of murine splenocytes was evaluated
to assess the immunomodulatory potential of rFip-fve.
The results clearly revealed that the specic immunomod-
ulatory activity of rFip-fve in Sf21 cells signicantly
exceeds that in bacterial cultures. Even though previous
experiments have shown that the glycosylation of rFip-fve
may not be essential to lymphocyte proliferative activity
(Ko et al. 1997), the present experimental result suggests
that the glycosylation region of Fip-fve may be critical in
enhancing and maintaining the required immunomodula-
tory activity of the T-lymphocyte. The rFip-fve expressed
in insect cells was processed and modied in a manner
1600
1400
1200
1000
800
600
400
200
m
I
L
-
2

c
o
n
c
e
n
t
r
a
t
i
o
n

(
p
g

m
l

1
)

0
ConA PBS rFip-fve
E. coli
rFip-fve
(extracellular)
sf21 cells
Figure 6 Effect of rFip-fve on release of IL-2 cytokine from murine
splenocytes. The immunomodulatory activity of rFip-fve expressed in
the culture supernatant of virus vAcP
10
SP
bbx
fve-infected Sf21 cells and
Escherichia coli was examined. The release of IL-2 cytokine from mur-
ine splenocytes was then examined. Data are expressed as relative
concentration obtained from ELISA reading under 540 nm, with refer-
ence to internal control. These data were obtained from three sepa-
rate experiments and are presented as mean SD.
Functional expression of Fip-fve C.-M. Wu et al.
1360 Journal compilation 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 13541362
2008 The Authors
more similar to that of its native counterpart than that in
bacterial cells. Therefore, the potential applications of
rFip-fve that is generated in Sf21 cells can be more effec-
tively evaluated that produced in E. coli.
Acknowledgement
The authors would like to thank the National Science
Council of the Republic of China, Taiwan, for nancially
supporting this research under Contract No. NSC 96-
2317-B-225-004.
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