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ORIGINAL ARTICLE

Journal of Applied Microbiology ISSN 1364-5072

Expression and purification 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

Keywords allergy, baculovirus insect cell expression system, Flammulina velutipes, fungal immunomodulatory protein, interleukin 2, nickel-affinity 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 efficient 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 purified from the supernatant of the infected Sf21 cells using a nickel-chelated affinity column, and was verified 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. Significance and Impact of the Study: The rFip-fve expressed in insect cells was processed and modified 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 .

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 purified 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 significantly 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

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Functional expression of Fip- fve

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 (61Æ 4%) 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-specific immune response, and efficiently 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 purification 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 purification of rFip-fve proteins. This study also is the first 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 26 C 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 (Met1–Thr115), was kindly provided by Dr Ko (from the Institute of Medical and Molecular Toxicology, Chung-shan Medical University, Taiwan). First, the Fve (Ser2–Thr114) fused with an additional

6His tag at the carboxyl terminus was obtained using a PCR with primers 5 ¢ -CG AGATCTTCCGCCACGTCGC- TC-3¢ ( Bgl II site underlined) and 5 ¢-AC GAATTCTAA- TGGTGATGGTGATGGTGAGTCTTCTTCCACTCAGC-3 ¢ ( Eco RI site underlined). The resulting PCR fragment was introduced at the Bgl II and Eco RI 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 Bgl II Bgl II restriction sites. Following treatment with endonucleases Bgl II, the generated Bgl II SP bbx DNA fragments were ligated to the Bgl II-digested pAcP 10 Fve, generating the recombinant plasmid pAcP 10 SP bbx Fve which is the SP bbx sequence in frame fused with: Fve–6His (Fig. 1). The recombinant plasmid was identified 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 0 Æ2 lg linear- ized AcRP23.LacZ DNA (PharMingen) using lipofectin. They were then purified 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 O’Reilly et al. (1992).

Expression of rFip-fve in baculovirus insect cell system

Sf21 cells were initially seeded into a cell culture flask (2 · 10 6 25 cm 2 flask) with approx. 80% confluence. 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 26 C. 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% confluence 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 26 C for 48, 72 and 96 h. At the end of infection, the cul- ture supernatants were harvested and then concentrated to a final 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

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f1 ori

Amp R ColE1 ori pAcP10-SP:fve 9719 bp polh gene p10 promoter polh promoter SPbbx Fip-fve
Amp R
ColE1 ori
pAcP10-SP:fve
9719 bp
polh gene
p10 promoter
polh promoter
SPbbx Fip-fve gene

Bg l II

EcoRI
EcoRI

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 1–4 amino acid of bombyxin protein (hatching) and 6His tag (solid line).

Innotech, San Leandro, CA). One microgram of purified 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 4 C 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 fluoride) membrane (Perkin-

Elmer, Wellesley, USA) in a Bio-Rad Trans-Blot system, following the manufacturer’s 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.

Purification of rFip-fve

6His-tagged rFip-fve protein from the culture supernatant of vAcP 10 SP bbx Fve-infected Sf21 cells was purified as described by van der Geld et al. (2002) with some modifi- cations. All purification steps were conducted at 4 C. 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 Tris–HCl and 60 mmol l ) 1 imidazole, pH 7 Æ9). 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 56 C 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 37 C with 0 Æ1 lg of TPCK (N-tosyl- l - phenylalanine chloro methyl ketone)-treated modified 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-flight mass spectrometer (Micromass Q-Tof Ultima, Taichung, Taiwan) at the Biotechnology Center of National Chung- Hsing University, Taiwan.

Expression and purification 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 purification, 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

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with 5 mmol l ) 1 reduced glutathione in 10 mmol l ) 1 Tris–HCl buffer (pH 8 Æ 0). After the GST:rFip-fve was treated with thrombin protease for 24 h at 22 C to release rFip-fve from the fusion protein.

Functional expression of Fip- fve

high-efficacy clone from six clones by immunoblot with anti-His antibody and determine the titer of stocks to be around 6 Æ9 · 10 7 plaque-forming units per ml (PFU ml ) 1 ) for the production of protein.

Measurement of IL-2 in mouse splenocytes

The biological activity of rFip-fve was measured as IL-2 was released from murine splenocytes. Briefly, splenocytes of mice (Balb C; 8–10 weeks old) were isolated by the Ficoll–Hypaque 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 7 Æ3. To 0 Æ 1 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 0 Æ1 ml of the RPMI 1640 medium was added to a final 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 37 C 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 streptavidin–horseradish peroxidase for 20 min, and then measured immediately at a wavelength of 450 nm using a microplate reader (TeKon Technologies, Irvine, CA).

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.

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 1–4 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 identified a

Purification and MALDI-MS identification of rFip-fve

The infected cell supernatant was collected for further purification using nickel-chelated affinity chromatography (NTA-Ni 2+ ) under native conditions, to purify the puta- tive rFip-fve expressed in Sf21 cells. The purified product exhibited a major band with an apparent molecular mass of 14 Æ 2 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 14 Æ2 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

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kDa

178

120

80

53

40

27

20

15

53

40

27

20

15

9

24 48 (h) Marker Uninfected Sf21 cells supernatant Infected Sf21 cells tsupernatan
24
48 (h)
Marker
Uninfected
Sf21 cells
supernatant
Infected
Sf21 cells
tsupernatan

Figure 2 SDS-PAGE and Western blot analysis of rFip-fve expression in Sf21 cells supernatant. Proteins (15 l g) 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.

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 14 Æ 2 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 verified by the MALDI-MS analysis (Fig. 4b). These results demonstrated that the purified 14 Æ 2 kDa protein was the mature rFip-fve and this

Marker

123

4

5

kDa

120

86

47

34

26

20

rFip-fve and this Marker 123 4 5 kDa 120 86 47 34 26 20 kDa 120

kDa

120

86

47

34

26

20

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 l g 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.

protein was correctly processed and secreted into the supernatant of infected Sf21 cells. Table 1 summarizes the purification of rFip-fve from insect cells. The rFip-fve recovery yield in one step using the NTA-Ni 2+ affinity column was nearly 62% and around 6 Æ25 mg purified rFip-fve was obtained from 1 l of culture supernatant – equivalent to 3 Æ1 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

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rFip-five

Purified

Marker

C.-M. Wu et al.

Functional expression of Fip- fve

(a)

(b)

kDa

53

40

27

20

15

9

expression of Fip- fve (a) (b) kDa 53 40 27 20 15 9 Figure 4 SDS-PAGE
expression of Fip- fve (a) (b) kDa 53 40 27 20 15 9 Figure 4 SDS-PAGE

Figure 4 SDS-PAGE and MALDI-MS analysis of purified rFip-fve. rFip- fve was purified by nickel-chelated affinity chromatography under native conditions. (a) The purity of the harvested rFip-fve was exam- ined on 15% SDS-PAGE gel. (b) Affinity-purified rFip-fve was sub- jected to protein identification by MALDI-MS analysis. Four peptide fragments of rFip-fve identified by MALDI-MS analysis are underlined.

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 14 Æ 2 kDa and then was compared with 1 lg purified 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 2 Æ 54, 4 Æ 26 and 6 Æ14 mg l ) 1 at 48 h, and 5 Æ6, 10 Æ14 and 6 Æ42 mg l ) 1 at 72 h, 4 Æ 21, 6 Æ62 and 2 Æ 04 mg l ) 1 at 96 h (Fig. 5). In this

Table 1 Purification of rFip-fve

Purification

Total protein

Total rFip-fve protein (mg)

Recovery

Purification

step

(mg)*

(%)

factor

Conditional

1140

10Æ 15

100

1

media

Ni-NTA

6 Æ 81

6 Æ 25

61Æ 6

103Æ 1

*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.

12 10 8 6 4 2 0 48 72 96 rFIP-fve protein production (mg l
12
10
8
6
4
2
0
48
72
96
rFIP-fve protein production (mg l –1 )

Time post-infection (h)

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.

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 purified 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 l g ml ) 1 ) of rFip- fve that had been purified from Sf21 insect cells and E. coli cells were 503 and 165 Æ3 pg ml ) 1 , respectively (Fig. 6). Evidently, the rFip-fve produced in Sf21 insect cells exhibited stronger specific 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 1234 Æ 8 pg ml ) 1 , which falls within the range 971 Æ 2–1498Æ 5 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 purified rFip-fve from Sf21 insect cells were 349 ± 50 Æ8, 503 ± 42 Æ1, 668 Æ1 ± 62 Æ 1 and 803 Æ 6 ± 42 Æ2 pg ml ) 1 , respectively (data not shown).

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1600 1400 1200 1000 800 600 400 200 0 mIL-2 concentration (pg ml –1 )
1600
1400
1200
1000
800
600
400
200
0
mIL-2 concentration (pg ml –1 )

ConA

PBS

rFip-fve

rFip-fve

 

(extracellular)

E. coli

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.

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 dose–response 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 purified procedure from the F. velutipes mushroom is rel- atively uneconomic, time-consuming and finally identified 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, purified 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 purified 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 purification (Choo et al. 2002). Accordingly, we inserted the synthetic SP bbx with the N-terminal 1–4 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 6 Æ25 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 efficiency 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 specific immunomod- ulatory activity of rFip-fve in Sf21 cells significantly 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 modified in a manner

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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 financially supporting this research under Contract No. NSC 96-

2317-B-225-004.

References

Choo, A.B., Dunn, R.D., Broady, K.W. and Raison, R.L. (2002) Soluble expression of a functional recombinant cytolytic immunotoxin in insect cells. Protein Expr Purif 24 , 338–347. Congote, L.F. and Li, Q. (1994) Accurate processing and secre- tion in the baculovirus expression system of an erythroid- cell-stimulating factor consisting of a chimaera of insulin- like growth II and an insect insulin-like peptide. Biochem J 299 , 101–107. Curtis, H., Sandoval, C., Oblin, C., Difalco, M.R. and Congote, L.F. (2002) Insect cell production of a secreted form of human alpha (1)-proteinase inhibitor as a bifunctional protein which inhibits neutrophil elastase and has growth factor-like activities. J Biotechnol 93 , 35–44. Dong, Y. and Shi, J.R. (2006) Application of primary culture technique to traditional Chinese medicine research. Zhong Xi Yi Jie He Xue Bao 4 , 90–93. Fiebich, B.L., Jager, B., Schollmann, C., Weindel, K., Wilting, J., Kochs, G., Marme, D., Hug, H. et al. (1993) Synthesis and assembly of functionally active human vascular endothelial growth factor homodimers in insect cells. Eur J Biochem 211 , 19–26. van der Geld, Y.M., Smook, M.L., Huitema, M.G., Harmsen, M.C., Limburg, P.C. and Kallenberg, C.G. (2002) Expression of recombinant proteinase 3, the autoantigen in Wegener’s granulomatosis, in insect cells. J Immunol Methods 264 , 195–205. Giraudo, C.L.C., Rumi, L. and Pasqualini, C.D. (1976) An improved method for PHA-culturing mouse lymphocytes using Ficoll–Hypaque gradient. Boll Ist Sieroter Milan 55 ,

304–307.

Groner, A. (1986) Specificity and safety of baculoviruses. In The Biology of Baculoviruses ed. Granados, R.R. and Federi- ci, B.A. p. 177 Boca Raton, FL: CRC Press. Haak-Frendscho, M., Kino, K., Sone, T. and Jardieu, P. (1993) Ling Zhi-8: a novel T cell mitogen induces cytokine pro- duction and upregulation of ICAM-1 expression. Cell Immunol 150 , 101–113. Hooker, A.D., Green, N.H., Baines, A.J., Bull, A.T., Jenkins, N., Strange, P.G. and James, D.C. (1999) Constraints on

Functional expression of Fip- fve

the transport and glycosylation of recombinant INF- c in Chinese hamster ovary and insect cells. Biotechnol Bioeng 63, 559–572. Hsieh, K.Y., Hsu, C.I., Lin, J.Y., Tsai, C.C. and Lin, R.H. (2003) Oral administration of an edible-mushroom- derived protein inhibits the development of food-allergic reactions in mice. Clin Exp Allergy 33 , 1595–1602. Hsieh, C.W., Lan, J.L., Meng, O., Cheng, Y.W., Huang, H.M. and Tsai, J.J. (2007) Eosinophil apoptosis induced by fungal immunomodulatory peptide-fve via reducing IL-5 alpha receptor. J Formos Med Assoc 106 , 36–43. Hsu, H.C., Hsu, C.I., Lin, R.H., Kao, C.L. and Lin, J.Y. (1997) Fip-vvo, a new fungal immunomodulatory protein isolated from Volvariella volvacea . Biochem J 323 , 557–565. Jarvis, D.L. (2003) Developing baculovirus-insect cell expres- sion systems for humanized recombinant glycoprotein pro- duction. Virology 310 , 1–7. Jinn, T.R., Wu, C.M., Tu, W.C. and Tzen Jason, T.C. (2006) Functional expression of FIP- gts , a fungal immunomodula- tory protein from Ganoderma tsugae in Sf21 insect cells. Biosci Biotechnol Biochem 70 , 2627–2634. Kato, T., Murata, T., Usui, T. and Park, E.Y. (2004) Improve- ment of the production of GFP uv - b 1,3-N-acetylglucosami- nyltransferase 2 fusion protein using a molecular chaperone-assisted insect-cell-based expression system. Bio- technol Bioeng 89 , 424–433. Kino, K., Yamashita, A., Yamaoka, K., Watanabe, J., Tanaka, S., Ko, K., Shimizu, K. and Tsunoo, H. (1989) Isolation

and characterization of a new immunomodulatory protein, ling zhi-8 (LZ-8), from Ganoderma lucidium . J Biol Chem 264 , 472–478. Ko, J.L., Hsu, C.I., Lin, R.H., Kao, C.L. and Lin, J.Y. (1995) A new fungal immunomodulatory protein, FIP-fve isolated from the edible mushroom, Flammulina velutipes and its complete amino acid sequence. Eur J Biochem 228 , 244–

249.

Ko, J.L., Lin, S.J., Hsu, C.I., Kao, C.L. and Lin, J.Y. (1997) Molecular cloning and expression of a fungal immuno-

modulatory protein, FIP-fve, from Flammulina velutipes.

J Formos Med Assoc 96 , 517–524.

Kondo, H., Ino, M., Suzuki, A., Ishizaki, H. and Iwami, M. (1996) Multiple gene copies for bombyxin, an insulin- related peptide of the silkmoth Bombyx mori : structural

signs for gene rearrangement and duplication responsible for generation of multiple molecular forms of bombyxin.

J Mol Biol 259 , 926–937.

Kost, T.A., Condreay, J.P. and Jarvis, D.L. (2005) Baculovirus

as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23, 567–575. Lin, W.H., Hung, C.H., Hsu, C.I. and Lin, J.Y. (1997) Dimer- ization of the N-terminal amphipathic alpha-helix domain of the fungal immunomodulatory protein from Ganoderma tsugae (Fip-gts) defined by a yeast two-hybrid system and site-directed mutagenesis. J Biol Chem 272, 20044–20048.

ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 1354–1362

1361

Functional expression of Fip-fve

Ma, P.W.K., Davis, T.R., Wood, H.A., Knipple, D.C. and Roe- lofs, W.L. (1998) Baculovirus expression of an insect gene that encodes multiple neuropeptides. Insect Biochem Mol Biol 28, 239–249. Mau, J.L., Lin, H.C. and Chen, C.C. (2002) Antioxidant prop- erties of several medicinal mushrooms. J Agric Food Chem 50, 6072–6077. Ng, T.B., Ngai, P.H. and Xia, L. (2006) An agglutinin with mitogenic and antiproliferative activities from the mush- room Flammulina velutipes . Mycologia 98 , 167–171. O’Reilly, D.R., Miller, L.K. and Luckow, V.A.(1992) Baculovi- rus Expression Vector: a Laboratory Manual. New York:

W.H. Freeman. Rajendra, W., Hackett, K.J., Buckley, E. and Hammock, B.D. (2006) Functional expression of lepidopteran-selective neu- rotoxin in baculovirus: potential for effective pest manage- ment. Biochim Biophys Acta 1760 , 158–163. Summers, M.D. and Smith, G.E.(1987) A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedure .

C.-M. Wu et al.

Texas Agricultural Experiment Station Bulletin No. 1555. Tanaka, S., Ko, K., Kino, K., Tsuchiya, K., Yamashita, A., Murasugi, A., Sakuma, S. and Tsunoo, H. (1989) Complete amino acid sequence of an immunomodulatory protein, ling zhi-8 (LZ-8). J Biol Chem 264 , 16372–

16377.

Wang, S.Y., Hsu, M.L., Hsu, H.C., Tzeng, C.H., Lee, S.S., Shiao, M.S. and Ho, C.K. (1997) The anti-tumor effect of Ganoderma lucidum is mediated by cytokines released from activated macrophages and T lymphocytes. Int J Cancer 70 , 699–705. Wasser, S.P. and Weis, A.L. (1999) Therapeutic effects of sub- stances occurring in higher Basidiomycetes mushrooms: a modern perspective. Crit Rev Immunol 19, 65–96. Wermerskirchen, A.S., LaTocha, D.H. and Clarke, B.L. (2000)

Adrenocorticotropic hormone controls Concanavalin A activation of rat lymphocytes by modulating IL-2 produc- tion. Life Sci 67 , 2177–2187.

1362

ª 2008 The Authors

Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 1354–1362