Expression and purification of recombinant swine interleukin-4

Comparative Immunology, Microbiology & Infectious Diseases 28 (2005) 17–35 www.elsevier.com/locate/cimid

Expression and purification of recombinant swine interleukin-4 A. Nuntapraserta, Y. Morib, K. Fujitaa, M. Yonedaa, R. Miuraa, K. Tsukiyama-Koharaa, C. Kaia,* a

Laboratory of Animal Research Center, Institution of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai Minato-ku, Tokyo 108-8639, Japan b National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan Accepted 16 March 2004

Abstract The swine interleukin-4 (SwIL-4) cDNA was cloned by RT-PCR. It was expressed using an expression vector pQE30 in E. coli, a baculovirus AcNPV vector pVL1392 in insect cells, and a pCAGGS vector in mammalian cells. The rSwIL-4 proteins expressed from bacteria and insect cells were purified using a chelating affinity column and a mAb-coupled immunoaffinity column. The amount of the products and their bioactivities were compared. All recombinant cytokines were efficiently reacted with the specific antibodies and the molecular weight of rSwIL-4 was approximately 16 kDa in E. coli, 15 and 18 kDa in insect cells, and 15 and 20 kDa in mammalian cells. Variations of molecular weight observed in insect and mammalian cells were probably due to different modification ways of glycosylation. All these recombinant proteins retained their antigenicity and were biologically active in inducing human TF-1 cell proliferation in vitro. The simple purification method will make it possible to evaluate the in vitro and in vivo effects of IL-4 in pigs. q 2004 Elsevier Ltd. All rights reserved. Keywords: Swine; Interleukin-4; Baculovirus expression; Bacterial expression; Mammalian expression

Re´sume´ Un clone de cADN de l’interleukine-4 porcine (SwIL-4) a e´te´ obtenue par l’RT-PCR. Elle s’est exprime´e sur E. coli avec le vecteur pQE30, sur les cellules pVL1392 d’insecte avec le vecteur vaculoviral AcNPV et sur les cellules mammaires avec le vecteur pCAGGS. Les prote´ines des SwIL-4 recombine´es (rSwIL-4) se sont exprime´es sur les cellules bacte´riennes ou d’insecte, et elles e´taient purifie´es utilisant unecolonne de che´late-affinite´ et d’immunoaffinite´ couple´ avec l’anticorps monoclonal. La quantite´ et la bioactivite´ des produits ont e´te´ compare´es. Toutes les cytokines recombine´es ont re´agi efficacement sur les anticorps spe´cifiques. Les poids mole´culaires e´taient * Corresponding author. Tel.: þ 81-3-5449-5497; fax: þ81-3-5449-5379. E-mail address: [email protected] (C. Kai). 0147-9571/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2004.03.012

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environ 16 kDa chez l’rSwIL de E. coli, 15 et 18 kDa chez celle d’insecte et 15 et 20 chez celle mammaires. Les variations en poids des produits d’insecte et mammaires e´taient probablement duesa` des manie`res varie´es de glycosylation. Les prote´ines recombine´es conservaient leur antigenicite´ et leur activite´ biologique d’induction de la prolife´ration de la cellule humaine TF-1. L’e´valuation des effets de l’IL-4 porcine in vitro et in vivo est possible avec cette me´thode de purification simple. q 2004 Elsevier Ltd. All rights reserved. Mots-cle´: Porc; Interleukine-4; Expression baculovirale; Expression bate´rienne; Expression mammaires

1. Introduction Interleukin-4 (IL-4) has been identified as an important mediator between inflammation and the immune response during the infections. IL-4 is a complex glycoprotein secreted by Th2 cell type response [1] and is able to influence Th cell differentiation [2] and B cells activation and proliferation [3]. IL-4 is a highly pleiotropic cytokine that interacts with various cells such as T cells, B cells, mast cells [4], thymocytes, hematopoietic cells, and fibroblasts [5]. IL-4 also involves in turning off the inflammatory response to prevent excessive inflammatory cytokine production and host damage, and promotes an antigenspecific humoral immune response. In contrast to other cytokines, effect of IL-4 is relatively restricted in terms of species specificity. Human IL-4 (HuIL-4) was reported to have the suppressive effect on the inflammatory activities on LPS-stimulated swine macrophages [6]. Although the biological function of IL-4 is well studied in mouse [7], rat [8], bovine [9, 10], canine [11] and human models [12], little is known about swine IL-4 (SwIL-4) and its activities due to the lack of specific detection system and biological activity assay system. SwIL-4 cDNA encodes 135 amino acids and has an amino acid sequence homology of 63 and 42% with the human and mouse species, respectively [13], however, there have been little reports describing its expression, preparation or its biological properties. IL-4 may be important in regulating the immune response in various diseases. This cytokine and its inhibitor might be significant as an indicator of infections or the therapeutic agents of inflammatory disease or cytokine adjuvant in control and prevention of pig diseases. To understand the biological role of IL-4 in swine diseases, it is necessary to characterize it in detail and to establish the method to quantitate circulating IL-4 level. In this study, we attempted the expression of rSwIL-4 in three kinds of recombinant expression systems and the one-step purifications of the bioactive rSwIL-4 from E. coli for use in the production of specific antibodies and from baculovirus system for use in basic studies as well as clinical applications of this cytokine. The biological activity of the recombinant protein on human TF-1 cells in vitro was also characterized.

2. Materials and methods 2.1. Construction of expression vectors Clone of SwIL-4 cDNA was multiplied by RT-PCR. Total RNA was isolated from ConA-stimulated swine PBMC. First-strand cDNAs were prepared from 1 mg of total

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Table 1 PCR primer sequences and conditions used for amplification of SwIL-4 sequences Vectors

Primer sequencea

Location of primer

pQE30b IL-4-Forward IL-4-Reverse

50 CG GGA TCC CAC AAG TGC GAC ATC ACC 30 50 CG GTC GAC TCA ACA CTT TGA GTA TTT 30

96–113 425–408

pVL1392c IL-4-Forward IL-4-Reverse

50 GAT CAG ATC TAT GGG TCT CAC C 30 50 CTA GGA ATT CTC AAC ACT TTG AG 30

24–35 425–413

pCAGGS IL-4-Forward IL-4-Reverse

50 ATG GGT CTC ACC TCC CAA CTG 30 50 TCA ACA CTT TGA GTA TTT 30

24–44 425–408

a PCR was performed in a volume of 50 ml (2.5 units Ampli Taq-polymerase, 25 mM MgCl2, 2.5 mM dNTPs, 10 mM primers), and the reaction was performed at 94 8C for 10 min, followed by 30 cycles of 94 8C for 1 min, 50 8C for 1 min, and 72 8C for 1 min, and then 72 8C for 10 min. b BamH I and Sal I restriction enzyme sites indicated by italics. c Bgl II and EcoR I restriction enzyme sites indicated by italics.

RNA using SuperScripte II reverse transcriptase (Gibco BRL, Gaithersburg, MD) and oligo (dT)15 primers (Promega, Madison, WI). The cDNA was amplified using Ampli Taq polymerase system (Perkin Elmer Applied Biosystems, Foster City, CA). Cloning was done following the basic protocols [14] and the manufacturer’s instructions. PCR primers with or without restriction enzyme sequences (Table 1) were designed based on the sequence termini of the SwIL-4 gene and the expression vectors used. Nucleotide sequence information was derived from Genbank accession number X68330 [13]. PCR products for bacterial and baculovirus expressions were digested with restriction enzymes and subcloned into each expression vectors. PCR product for mammalian expression was introduced into PCR-TA cloning vector (pCR2.1, Invitrogen, San Diego, CA) and sequenced. The rSwIL-4-specific insert was then cut out from TA vector using restriction enzymes. The DNA inserts were gel-purified using QIAEXw II gel extraction kit (Qiagen, Hilden, Germany). The expression vectors were also digested by restriction enzymes and treated with calf intestinal alkaline phosphatase. Ligation of SwIL-4 insert and vector was done overnight (14 8C) using T4-DNA ligase (Promega). Transformation was performed according to standard protocol [14] using heat shock technique and competent E. coli DH5a cells (Toyobo Co., Ltd, Osaka, Japan). Bacterial colonies were selected on agar plates using ampicillin (100 mg/ml). Single clone was picked up, and its nucleotide sequence was analyzed using an ABI Prism 377 sequencer (Applied Biosystems) and data analysis software (GENETYX-MAC ver.10, Software Development, Tokyo, Japan). 2.2. Expression and purification of rSwIL-4 in bacterial system For bacterial expression, we used a vector (pQE30, Qiagen, Hilden, Germany) coding for a N-terminal detection and purification epitope (6 £ His-tagged) as specified by the manufacturer. Accordingly, the SwIL-4 secretion signal at 50 was not incorporated in PCR

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product. The pQE30-SwIL-4 cloned from E. coli strain M15 (Qiagen, Hilden, Germany) was grown at 37 8C in 1 l of Luria-bertani (LB) medium (100 mg/ml ampicillin and 25 mg/ml kanamycin) to the optimal density of 0.6 (600 nm). Then, IPTG (2 mM) was added to induce protein production for 4 h at 30 8C. The cells were harvested by centrifugation at 8000 rpm for 10 min at 4 8C, dissolved in lysis buffer (25 mM Tris-HCl, pH 7.4 containing 5 mM MgCl2, 1% Triton-X-100, 1% N-lauroyl-sarcosine, and 10 mM imidazole), and incubated on ice for 1 h. The suspension was sonicated on ice and centrifuged at 10,000 rpm at 4 8C for 30 min to remove insoluble materials. Expression of recombinant protein was detected by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting using the anti-RGS-6 £ His mAb (Qiagen, Hilden, Germany) as described below. Large-scale expression revealed that rSwIL-4 was produced in soluble protein. The soluble fraction of the protein was extensively dialyzed in loading buffer (25 mM Tris-HCl, pH 7.4, 500 mM NaCl and 10 mM imidazole) overnight. Protein was purified using HiTrap chelating affinity column (Amersham Biosciences, APB, Uppsala, Sweden). The column was equilibrated with 0.1 M NiSO4 £ 6H2O in loading buffer, loaded solubilized lysate, and washed. The recombinant proteins were then eluted with elution buffer in various concentrations of imidazole (100, 200, 300, 500 mM, and 1 M). The purified protein was resolved on 12.5% SDS-PAGE, stained by 0.5% Coomassie Brilliant Blue-R250 (CBB) and analyzed by western blotting using the rabbit anti-SwIL-4 antibody as described below. The eluted protein fractions of rSwIL-4 (positive by western blotting analysis) were pooled and concentrated using Biomax-10 Ultrafree-10 concentrators (Millipore Corp., Bedford, MA). This rSwIL-4 was dialyzed in phosphate buffered saline (PBS) overnight and sterilized by filtration for the use as an immunogen and in a bioassay. The purified rSwIL-4 was confirmed its IL-4 protein by western blotting analysis using rabbit anti-HuIL-4 polyclonal antibody (Southern Biotechnology Associated, Inc., Birmingham, AL) as described below. HuIL-4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) protein was used as a positive control. The concentration of active SwIL-4 was determined by sandwich ELISA as described below. 2.3. Anti-SwIL-4 antibodies and sandwich ELISA for SwIL-4 We produced specific anti-SwIL-4 antibodies, and established the sandwich ELISA for swine IL-4 (Nuntaprasert et al., submitted). The anti-SwIL-4 monoclonal (3 clones of 1604-6, 2123-3-3, and 270-4-6) and polyclonal antibodies from rabbit and goat were produced and used for detecting rSwIL-4 in immunoprecipitation, western blotting analysis, and intracellular staining. The mAb clone 160-4-6 was used for immunoaffinity purfication of rSwIL-4 produced from baculovirus system. A sandwich ELISA for SwIL-4 was used for measuring the concentration of active SwIL-4. 2.4. Expression and purification of rSwIL-4 in the baculovirus system The rSwIL-4 was expressed in an insect cell line that is specialized for secretion of protein under serum-free conditions. The SwIL-4 gene including the specific signal and restriction enzyme sequences was amplified by PCR. The purified PCR products were then

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ligated into the transfer vector pVL1392 (Pharmingen, San Diego, CA). Linearized BaculoGold DNA (Pharmigen) was cotransfected with the recombinant plasmid (pVL1392-SwIL-4) into the Spodoptera frugiperda cell line SF21AE in serum-free SF-900II SFM medium (Gibco BRL, Life Technologies, Inc., Rockville, MD) using Lipofectin (Gibco BRL, Life Technologies, Inc.) as described by the manufacturer. Three days after transfection, the culture supernatants containing the recombinant virus (AcpVL1392-SwIL-4) were harvested and subjected to standard plaque purification methods [15]. Recombinant virus at a multiplicity of infection (MOI) of 1 plaque forming units was used to infect the Trichoplusia ni-derived cell line BTI TN 5B1-4 (Tn5) for the recombinant protein production. A large-scale (1 l) production of rSwIL-4 was performed using serum free Express Five medium (Gibco BRL, Life Technologies, Inc.). Four days after infection at 27 8C, the medium supernatants and disrupted cells were harvested and stored at 2 80 8C until analysis. Expression of recombinant protein was determined by indirect immunofluorescence (IF) assay and western blotting analysis using anti-SwIL-4 mAb (212-3-3) as described below. Baculovirus expressed proteins were purified by immunoaffinity chromatography. The supernatant of Tn5 cells infected with AcpVL1392-SwIL-4 was harvested at 4 dpi, concentrated by filtration with Sartocon Micro polysulfone ultrafilter (Sartorius AG, Go¨ttingen, Germany), removed baculovirus particles by using Ultrafree-15 centrifugal filter device (Biomox-100, Millipore Corp.), and used for further purification. The purification was performed on an immunoaffinity chromatography column immobilized with anti-SwIL-4 mAb (160-4-6). Briefly, the HiTrap Protein-A affinity column (2 ml, Amersham Biosciences) after mAb binding at the concentration of 20 mg/ml was covalently crosslinked with immediate crosslinker DMP (Dimethylpimelimidate, Pierce, Chemical Co. Rockford, IL) and blocked of remaining active sites. Two hundred milliliter of the concentrated supernatants diluted with 10 mM Tris-HCl buffer (pH 7.5) were applied to the immobilized mAb column and incubated for 6 h at room temperature. After applying the supernatants, the column was washed with 10 mM Tris-HCl buffer (pH 7.5) and then eluted with 100 mM Glycine-HCl (pH 2.8). Subsequently, the eluted solution was quickly adjusted to neutral pH by adding 1 M Tris-HCl (pH 9.0). Fractions of purified rSwIL-4 were collected, analyzed by western blotting analysis using anti-SwIL-4 mAb (270-4-6) as described below, pooled, concentrated and dialyzed against PBS at 4 8C. The preparation was stored at 2 80 8C until analysis of biological activity. The active IL-4 concentration was determined using sandwich ELISA system. 2.5. Transient expression of rSwIL-4 in the mammalian cell In order to produce recombinant protein in COS-7 (SV40 transformed African green monkey kidney; ATCC, Rockville, MD) cells, SwIL-4 cDNA including signal sequence was subcloned into pCAGGS vector under the control of the CAG promoter. COS-7 cells were transiently transfected with pCAGGS-SwIL-4 using FuGENE 6 reagent (Boehringer Mannheim, GmbH, Mannheim, Germany) according to the manufacturer’s protocols. The pCAGGS vector was used as a mock transfection. The cells and supernatants obtained from COS-7 cells transiently transfected with SwIL-4 were harvested at 24 h post transfection and filtered through a 0.22 mM filter. The supernatants (20 ml) were

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size-fractionated by ultrafiltration (30 kDa . MW . 10 kDa), concentrated 10-fold using Microcon YM-10 microconcentrators (Millipore, Bedford, HA) to enrich rSwIL-4 and used for bioassay. The expressed gene and protein was determined using RT-PCR, immunoprecipitation, and intracellular staining as described below. The concentration of active SwIL-4 was determined by sandwich ELISA. RT-PCR was performed at 24 h after transfection. Briefly, total RNA was isolated from 1 £ 106 cells using the ISOGEN (Nippon Gene, Tokyo, Japan) and then transcribed by first strand reverse transcriptase reaction using oligo (dT)15 primers (Promega, Madison, WI) and SuperScript II reverse transcriptase (Gibco BRL, Life Technologies, Inc.), following manufacturer’s instructions. The cDNA of transfected cells was used as a template in subsequent PCR amplification for the rSwIL-4 (Table 1). The PCR reaction was carried out in a Thermal cycler (Perkin Elmer) and the PCR products were separated in 1% of agarose gels containing 0.005% ethidium bromide (Sigma-Aldrich, Chicago, IL). Intracellular staining was analyzed on COS-7 cells at 24 h after transfection by flow cytometry. Briefly, a total of 1 £ 106 transfected COS-7 cells were washed, fixed with 3% paraformaldehyde, incubated with a specific antibody of anti-SwIL-4 mAb (270-4-6, 1:500 dilution), stained with FITC conjugated goat anti-mouse IgG (Caltag Laboratories, CA) (1:1000 dilution), and then evaluated by flow cytometry using FACScan (Becton Dickinson, Valley View, CA). 2.6. Immunoprecipitation and western blotting analysis Immunoprecipitation was used to detect secreted rSwIL-4 protein from COS-7 cell culture medium. Briefly, the concentrated supernatant (2 ml) was incubated with 1 mg of the purified anti-SwIL-4 mAb (270-4-6) overnight at 4 8C with rotating. Twenty microliters of 50% slurry of Protein-A sepharose beads (Amersham Biosciences) was then added for 2 h to collect the immune complexes. These complexes were then washed three times at 4 8C with lysis buffer (1% Triton X-100, 0.1% SDS, 0.01% (v/v) NP-40, 2 mg/ml aprotinin, 100 mg/ml PMSF, 0.15 M NaCl, 2 mM EDTA, and 10 mM sodium phosphate pH 7.2), vortexed after resuspension, and pelleted at 10,000 rpm for 1 min between washes. The beads were then rinsed with TN buffer (50 mM Tris-HCl pH 7.2, 0.15 M NaCl), centrifuged, and then resuspended in SDS-PAGE sample buffer. After heated at 100 8C for 5 min, samples were separated on 15% SDS-PAGE and western blotting was performed using the goat anti-SwIL-4 polyclonal antibody. For western blotting analysis, the rSwIL-4 (E. coli), rSwIL-4 (insect cells), and rHuIL-4 (E. coli) proteins were loaded at 100 ng/lane. The lysate of COS-7 cells (10 transiently transfected with plasmid encoding SwIL-4 after immunoprecipitation was loaded at 20 ml/lane. Proteins were separated by 12.5 or 15% SDS-PAGE according to Laemmli [16] and transferred to polyvinylidene difluoride (PVDF) membrances (Amersham Biosciences). The anti-SwIL-4 antibodies (mAb and polyclonal antibodies) or anti-HuIL-4 antibody were used at 1:1000 dilution and immunodetection was performed with appropriate HRP-conjugated secondary antibodies and the positive band was visualized by 3, 30 -diaminobenzidine tetrahydrochloride (DAB; Dojindo Laboratories, Kumamoto, Japan) staining.

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2.7. The glycosylation analysis of expressed rSwIL-4 Deglycosylation was assessed by addition of different concentrations of tunicamycin (Sigma-Aldrich, at the doses of 5, 10, and 50 mg/ml) to the infected Tn5 cells with AcpVL1392-SwIL-4 for 4 days prior to analysis on 12.5% of SDS-PAGE gels and western blotting using the anti-SwIL-4 mAb (160-4-6) as above. 2.8. Indirect immunofluorescence (IF) assay In order to perform the indirect IF assay, the SF21AE cells were cultured for 3 days after infection with control wild type AcNPV or AcpVL1392-SwIL-4, harvested, and washed twice with PBS. The cells were smeared on glass slides, incubated with an anti-SwIL-4 mAb (270-4-6) for 30 min at 37 8C, and washed with PBS. Then the cells were incubated with the goat anti-mouse-IgG antibody conjugated with fluorescein isothiocyanate (FITC; Cappel, Aurora, OH). The stained cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan). 2.9. The in vitro biological activity of rSwIL-4 We performed the proliferation assay using human TF-1 cell line in which proliferations were induced by human IL-4 (HuIL-4), with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) colorimetric method as described [17,18]. Briefly, TF-1 cells, which were mixed with a series of successive dilution of the rSwIL-4 (0.005 – 20 ng/ml) samples, were seeded at 2 £ 105 cells/ml in 96-well microplate. At 48 h after incubation at 37 8C in 5% CO2, MTT was added and incubated for an additional 2 h, formazan crystals were solubilized and the optical density at 570 nm were determined. The units per mg of rSwIL-4 activities were determined by calculation from the standard curve generated using rHuIL-4 as a standard (Strathmann Biotech Gmbh, Germany, 2.5 £ 104 units/ml) and the IL-4 concentration giving half-maximum proliferation being defined as 1 unit.

3. Results 3.1. Cloning of rSwIL-4 cDNA The rSwIL-4 cDNA was cloned by RT-PCR from activated swine PBMC. The rSwIL-4 gene without signal sequence was around 350 bp (data not shown) and subcloned into E. coli expression vector. The rSwIL-4 gene with secreting signal sequence was around 404 bp (data not shown) and subcloned into baculovirus and mammalian expression vector. These amplified cDNA fragments were subcloned into pQE30-SwIL-4, pVL1392SwIL-4, and pCAGGS-SwIL-4 vectors for the expression of recombinant protein in bacteria, baculovirus and mammalian expression systems, respectively. Compared with the nucleotide sequence data reported previously (the accession number is X68330),

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nucleotide sequences of these clones had no differences or mutation in the sequences of those resulting plasmids (data not shown). 3.2. Bacterial expression and purification The rSwIL-4 protein was expressed in bacteria with polyhistidine (6 £ His-tagged) affinity tag and was purified using the 6 £ His-tagged-based nickel HiTrap chelating chromatography as described in materials and methods. Under culture condition, the rSwIL-4 was expressed as a relative molecular weight of approximately 16 kDa in 15% SDS-PAGE stained with CBB (Fig. 1(A)) and western blotting analysis using the anti6 £ His mAb (Fig. 1(B)). To obtain the purified rSwIL-4, we used a single-step affinity purification procedure. The soluble extract from E. coli was directly loaded and purified onto the 6 £ His-tagged-based nickel HiTrap chelating column. Following extensive washing, the bound proteins were eluted with 100, 200, 300, 500 mM, and 1 M imidazole, and the fractions containing pure protein was found in elution buffer with 200 mM imidazole. The purity of recombinant protein was confirmed by a homogeneous banding of 16 kDa when analyzed by 12.5% SDS-PAGE stained with CBB (Fig. 1(C)). The purified rSwIL-4 was used for the production of anti-SwIL-4 antibodies. By western blotting using the rabbit anti-SwIL-4 polyclonal antibody, the major band of 16 kDa was recognized (Fig. 1(D)). A single peak could be observed when analyzed on AKTAprime Liquid chromatography (data not shown). We confirmed the purified rSwIL-4 from bacteria by western blot analysis using the rabbit ani-HuIL-4 polyclonal antibody (Fig. 4(A)). A summary of the purification procedure for the rSwIL-4 produced from bacteria is shown in Table 2. A purification of more than 60-fold increase of activity with a yield of about 50% was achieved. In this study, the overall yield of concentrated purified rSwIL-4 from 1 l of bacterial culture was 0.4 mg of rSwIL-4. 3.3. Baculovirus expression and purification To obtain rSwIL-4 with the baculovirus expression system, recombinant transfer vector pVL1392-SwIL-4 was constructed and co-transfected with baculovirus DNA. After confirmation of plaque purification, three putative recombinants were obtained. The SF21AE cells were infected with AcpVL1392-SwIL-4 at MOI of 1.0 for 3 days, and the expression of secreted rSwIL-4 on the cell surface was examined by the indirect IF assay with the anti-SwIL-4 mAb (270-4-6) (Fig. 2(A)). The control AcNPV did not show any fluorescence, therefore, the rSwIL-4 was expressed on the surface of AcpVL1392-SwIL-4 infected cells. When Tn5 cells were infected with the same recombinants at MOI of 1.0, a various sizes of proteins were secreted and accumulated in the culture supernatants from 48 h post infection. Western blotting analysis (Fig. 2(B)) with the anti-SwIL-4 mAb (clone 212-3-3) revealed that the expressed rSwIL-4 consisted of secreted proteins in two different sizes of about 15 and 18 kDa in the medium, which were not detected in mockinfected or infected with control baculovirus (data not shown). Supernatants from the baculovirus-infected Tn5 cells were harvested after 96 h, which had been shown to be a suitable time-point for the highest protein production in preliminary studies. The maximum concentration created by large-scale production of insect cell (Tn5) supernatant

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Fig. 1. Expression and purification of the 6 £ His-tagged rSwIL-4 protein were performed. (A) The expressed rSwIL-4 in E. coli M15 were separated on 15% SDS-PAGE and stained with CBB and (B) western blotting analysis using anti-RGS-6 £ His mAb. Purification was performed using the HiTrap affinity column. Each fractions from purification step were subjected to analysis on 12.5% SDS-PAGE. Gels were stained with (C) CBB and (D) western blotting with the rabbit anti-SwIL-4 polyclonal antibody. M, Size markers (kDa); U, total protein before IPTG induction; I, total protein after IPTG (2 mM) induction; S, soluble fraction from induced cells; P, insoluble fraction from induced cells; F, flow through fraction; W, washing buffer fraction; lane E1–E5, each fraction eluted by 100, 200, 300, 500 mM, and 1 M of imidazole contained the recombinant protein. The arrowheads indicate the recombinant proteins.

contained 10 mg/ml of rSwIL-4 (Table 2). Preliminary experiments indicated that culture medium could be used as the source of purification. We used the serum-free express five culture medium, because it has a low contaminating protein contents. This culture medium contained approximately 30% of secreted protein and was a preferable source for purification of rSwIL-4 protein. The one-step immunoaffinity purification as described in Materials and Methods was used to obtain pure rSwIL-4. The mAb clone 160-4-6 directed against rSwIL-4 was immobilized to Protein-A column. Most rSwIL-4 was bound to the column under these conditions. The elution of rSwIL-4 was optimal when using 100 mM

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Purification step

Bacteria Cell pellet sonicated Elute pooled peak from HiTrap column Concentrated Insect cells Original medium Media concentrated Elute pooled peak from immunoaffinity column Concentrated

Volume (ml)

Activitya £ 104 (units/ml)

Total activity £ 104 (units)

SwIL-4b (mg/ml)

Total SwIL-4 (mg)

Specific activity £ 104 (units/mgIL-4)

Yield (%)

Fold

1 50

100 30

1 2

100 60

0.50 0.02

50 0.6

2 100

100 60

10

5

50

0.04

0.4

125

50

62.5

3750 2400 600

0.01 0.03 0.02

10 6 0.3

375 400 2000

100 64 16

1 1.1 5.3

500

0.04

0.2

2500

13

6.7

10 8

001 0.005

0.02 0.01

500 800

100 80

1 1.6

1000 200 15 5

3.75 12 40 100

c

COS-7 cells Original medium Media concentrated a

20 2

0.5 4

Biological activity was analyzed by mouse TF-1 cells proliferation test. The units/ml was calculated by comparison with the activity of rHuIL-4 as a standard (2.5 £ 104 units/ml) (Fig. 5) and converted into total activity (units) by the following equation: total activity (units) ¼ biological activity (units/ml) £ total volume (ml). b The amount of rSwIL-4 in whole protein preparations was performed using a sandwich ELISA assay as described in materials and methods. c Secreted rSwIL-4 from supernatants of mammalian cells was not performed purification.

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Table 2 Analytical data of the expression or purification of rSwIL-4 protein from three kinds of expression systems

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Fig. 2. The indirect immunofluorescence assay using the anti-SwIL-4 mAb (270-4-6). (A) Three days after infection SF21AE cells infected with recombinant AcpVL1392-SwIL-4 (rSwIL-4) showed significant fluorescence whereas control wild type AcNPV (control) did not show positive fluorescence. (B) The accumulation of recombinant protein in AcpVL1392-SwIL-4-infected cell culture supernatants was analyzed by time course. (C) The amount of recombinant proteins was analyzed in 12.5% SDS-PAGE and western blotting using anti-SwIL-4 mAb (212-3-3). Purification of rSwIL-4 produced from insect cells (4 days after culture) by the immunoaffinity column using the anti-SwIL-4 mAb (160-4-6). Fractions from each step of the purification were subjected to analysis on 12.5% SDS-PAGE. Gels were stained with western blotting using the anti-SwIL-4 mAb (270-4-6). M, size markers (kDa); P and S, cells and supernatant of AcpVL1392-SwIL-4 infected cells at indicate days after infection; F, flow through fraction; W, washing buffer fraction; E1–E5, eluted fractions contained recombinant proteins. The specific bands of 15 and 18 kDa are indicated by arrowhead.

Glycine-HCl pH 2.8 and led to pure rSwIL-4 protein. This purified proteins migrated as two bands with approximately 15 and 18 kDa on 12.5% SDS-PAGE and western blotting analysis using the anti-SwIL-4 mAb (270-4-6) (Fig. 2(C)). In Table 2, the final concentration of concentrated rSwIL-4 was enriched about 7-fold increase of activity,

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yielding a specific activity of 2.5 £ 107 units/mg, and a total recovery of 13% because much rSwIL-4 remained bound to the column (as indicated in the lane with the column material after elution). 3.4. Mammalian expression Using a transient mammalian expression system, we expressed rSwIL-4. The expression and secretion of this protein from COS-7 cells was detected at 24 h after transfection. Transcription of SwIL-4 mRNA was evaluated by RT-PCR amplification from transfected cells. RT-PCR products amplified from RNA of pCAGGS-SwIL-4 transfected cells were revealed as the expected products (404 bp), confirming correct transcription (Fig. 3(A)). Western blotting after immunoprecipitation analysis also confirmed that this recombinant protein still retained its antigenicity and could be recognized by the goat anti-SwIL-4 polyclonal antibody. Secreted rSwIL-4 was produced in two different sizes of 15 and 20 kDa (Fig. 3(B)). Transfected COS-7 cells were tested for protein expression by intracellular staining using the anti-SwIL-4 mAb (160-4-6) (Fig. 3(C)). Approximately, 40% of the transfected COS-7 cells expressed rSwIL-4. The concentration of secreted rSwIL-4 protein in 2 ml-concentrated supernatant as described in Materials and Methods was approximately 0.005 mg/ml at 24 h post infection, yielding a specific activity of 8 £ 106 units/mg (Table 2). This activity concentration was considered to be sufficient for further biological testing. 3.5. Molecular weights of rSwIL4 The biochemical features of rSwIL-4 were characterized. The predicted molecular weight of SwIL-4 deduced from the amino acid sequence is 15 kDa, which was the expected size before removal of signal peptide and secretion. Western blotting analysis with the anti-SwIL-4 mAb or the polyclonal antibody revealed that the bacterial fusion product had an apparent molecular weight of 16 kDa whereas the baculovirus and mammalian cell products had higher molecular weights than those predicted. The insect cell-derived rSwIL-4 migrated into two different sizes of 15 and 18 kDa and mammalian cell-derived rSwIL-4 migrated into 15 and 20 kDa The glycosylation process in different cells may reflect the gradual increase in molecular mass. Tunicamycin (5 and 10 mg/ml), an inhibitor of N-glycosylation, added for 4 days prior to analysis to Tn5 insect cells caused an apparent decrease of 18 kDa and these concentrations were sufficient to inhibit the glycosylated form of rSwIL-4 in culture supernatant completely as compared with the non-treated supernatant (Fig. 4(B)). The intensity of the 15 kDa band was increased after tunicamycin treatment, suggesting that this protein is unglycosylated form. The results demonstrated that the heterogeneity in migrated molecules in SDS-PAGE analysis might be due to differences in glycosylation. 3.6. Biological activity of rSwIL-4 Biological activity of rSwIL-4 was analyzed using the human cell line TF-1, which proliferates in response to rHuIL-4. The TF-1 cells showed proliferation in

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Fig. 3. In vitro transient expression of rSwIL-4 in COS-7 cells at 24 h after transfection. (A) RT-PCR analysis of IL-4 gene expression in COS-7 cells was performed as described in materials and methods. (B) The proteins in culture supernatant of transfected cells were immunoprecipitated with the anti-SwIL-4 mAb (270-4-6) and analyzed by western blotting using the goat anti-SwIL-4 polyclonal antibody. (C) At 24 h after transfection, the labeled cells were monitored by flow cytometry using the anti-SwIL-4 mAb (160-4-6). The histogram represents the intensity of fluorescence in the FL1 channel after staining with the anti-SwIL-4 mAb. M, size markers (bp); 1, the absence of RT-PCR from non-transfected cells (no plasmid); 2, the presence of RT-PCR from non-transfected cells (cDNA of COS-7 cells); 3, the control represents plasmid DNA containing cDNA of SwIL-4; 4, cDNA of COS-7 cells transfected with pCAGGS-SwIL-4; 5, total RNA of COS-7 cells transfected with pCAGGS-SwIL-4; mock, mock-transfected COS-7 cells; IL-4, COS-7 cells transfected with rSwIL-4. The arrows indicate the band of SwIL-4 gene and arrowheads indicate the secreted proteins from supernatant.

a dose-dependent manner after the stimulation with either bacteria or insect cell produced rSwIL-4 as well as rHuIL-4 (Fig. 5). The logarithmic phase of the growth curve is parallel with HuIL-4, confirming that the rSwIL-4 possesses analogous function [19]. Thus, the rSwIL-4 proteins were biologically functional, and glycosylation of recombinant proteins may implicate in higher biological activity. This assay gives 50% of maximal responses induced by approximately 0.1 ng/ml of rSwIL-4 from insect cells whereas by 2 ng/ml of rHuIL-4. The TF-1 cells are approximately 10-fold more responsive to rSwIL-4

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Fig. 4. (A) Cross-reactivity of the rabbit anti-HuIL-4 IgG antibody against rSwIL-4 produced from bacteria by western blotting analysis. M, size markers (kDa); B, affinity purified rSwIL-4 from bacteria; H, rHuIL-4 produced from bacteria. The arrows indicate the rSwIL-4 proteins. (B) Effect of different tunicamycin concentrations on the glycosylation of rSwIL-4 from insect cell supernatant at 4 days after infection was analyzed by western blotting with the anti-SwIL-4 mAb (160-4-6). M, Size markers (kDa); P and S, cells and supernatant of AcpVL1392SwIL-4 infected cells with tunicamycin at indicated doses. The arrows indicate the rSwIL-4 proteins.

Fig. 5. Comparison of TF-1 human cell responses to each of rSwIL-4. TF-1 cells proliferated after stimulation of rSwIL-4 purified from bacteria (W) and from insect cells (X) in a dose-dependent manner. The rHuIL-4 produced from bacteria (B) was used as a control. The units/ml activity was calculated by comparing with the activity of rHuIL-4 (2.5 £ 104 units/ml) as a standard.

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than rHuIL-4. Table 2 indicates a summary of the specific activity of rSwIL-4 preparations after each purification steps. We estimated the specific activity of rSwIL-4 preparation by measuring the amounts of IL-4 content using a sandwich ELISA [20]. On the basis of bioassay, the total units and units/mg of purified rSwIL-4 from insect cell preparation were higher than those of bacteria. The specific activity of the concentrated sample from bacteria after the affinity column step was approximately 1.3 £ 106 units/mg of rSwIL-4 protein with a 50% recovery and approximately 60-fold increase of activities. The specific activity of the concentrated sample from insect cells after the affinity column step was approximately 2.5 £ 107 units/mg of rSwIL-4 protein with a 13% recovery and approximately 7-fold increase of activities. The enriched secretion from COS-7 cell preparation had tendency to show the highest value (approximately 8 £ 106 units/mg of rSwIL-4). However, we did not perform further purification step of this mammalian preparation because the production of rSwIL-4 in COS-7 cells was much lower than that of insect cells.

4. Discussion In this study, we cloned the SwIL-4 cDNA from swine PBMC and developed three different kinds of expression vectors (pQE30, pVL1392, and pCAGGS). We compared the expression level, efficiency of purification, and biological activities of each rSwIL-4 produced from bacteria, insect cells (Tn5) and mammalian cells (COS-7). We described a simple purification procedure that allowed the preparation of rSwIL-4 from bacteria and from insect cell supernatant in a one-step purification protocol. The simplicity of the process could make this approach for the routine purification of the rSwIL-4. The production of the rSwIL-4 in bacteria was our initial effort to produce specific antibody and to establish immunoassay reagents for detection of swine IL-4 in biological samples. This data clearly demonstrated that all attempts to express rSwIL-4 using the protein expression vector pQE30 in E. coli, baculovirus pVL1392 in insect cells, and pCAGGS vector in mammalian cells were successful. The mature SwIL-4 has a predicted molecular mass of 15 kDa from its sequence. Considering the putative signal sequence, the size of the mature recombinant protein produced in this study is 182 amino acid residues of 16 kDa. We found that the relative molecular weight of recombinant protein was slightly larger size compared to its calculation based upon translation of the cDNA sequence [13]. The 6 £ His-tagged rSwIL-4 could be expressed as soluble form by an E. coli expression vector and purified using one-step affinity chromatography with HiTrap chelating affinity column. The increased size may be due to 6 £ His peptide tag present in the protein or another modification to the proteins or variably processed of precursor protein. Both constructs from eukaryotic expression systems (insect and mammalian cells) contained a signal peptide that resulted in the secretion of the expressed recombinant proteins in the media. Western blotting analysis of the secreted rSwIL-4 produced from insect cells (Fig. 2(B)) and COS-7 cells (Fig. 3(B)) showed that these proteins migrated ranging from 18 to 20 kDa as the larger band than the predicted mature protein size based on its peptide sequence, which suggested that

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the molecule is glycosylated. The secreted ovine IL-4 from baculovirus expression system had a molecular mass of 17 kDa [21]. Natural glycosylated IL-4 has been reported at a molecular weight of 20 kDa in human [22] and 19 kDa in mouse [23]. These apparent heterogeneities in size are probably caused by the biochemical modification or differences in glycosylation of rSwIL-4 molecule after being processed in insect cells or in COS-7 cells. As the predicted amino acid sequence at positions 61, 96, and 102 display the three potential N-glycosylation sites (Asn-X-Ser/Thr) in the extracellular domain, N-glycosylation might explain the larger molecular size of the expressed rSwIL-4. The contribution of glycosylation was examined by treatment with tunicamycin, and we found that rSwIL-4 molecule showed at least one glycosylated form. The 15 kDa band probably corresponds to non-glycosylated, and the 18 kDa band might be a glycosylated or a glycosylated precursor protein [24]. We have examined the production of secreted rSwIL-4 with baculovirus system. The amount of secreted rSwIL-4 in culture supernatant of infected cells increased and reached a plateau at 96 h post infection. The supernatants contained approximately 10 mg/ml of rSwIL-4 with a biological activity of 3.8 £ 106 units/mg as assessed on TF-1 cells response. This result indicated that this bioactive rSwIL-4 expressed in baculovirus system could be used directly (without further purification) for routine lymphocyte culture or other immunological experiments. The rSwIL-4 could be purified from supernatant of insect cells using anti-SwIL-4 mAb-coupled immunoaffinity column chromatography. The specific activity after the last purification step was increased 7-fold and the amount was 13% recovery from the starting material. Compared with E. coli, the mammalian COS-7 cells can express recombinant proteins with proper folding and correct glycosylation. However, since the transient expression level in the COS-7 cells was low (1 mg/ml) in the culture medium, the purification of the rSwIL-4 from this system would be a formidable task. Higher yield may be obtained by production of permanent SwIL-4 transfected cell line containing introduced antibiotic resistance gene such as neomycin analog (G418). In this study, the rSwIL-4 produced from bacteria is often unable to be properly folded, and those recombinants from mammalian cells (COS-7) were obtained only at a few amount. Therefore, the insect cell system was likely to be more appropriate than the bacteria and the mammalian systems to express enough amount of rSwIL-4 for further in vitro and in vivo studies. Using this system, we generated and purified 200 mg quantities of essential rSwIL-4 protein from 1000 ml of insect cell culture. The IL-4 biological activity can be measured and confirmed by the proliferation assays using many specific cell lines such as mouse MC/9 cells for mouse IL-4 [25], the intestine endotheial cell line IEC-6 for rat IL-4 [26], and human TF-1 cells for human IL-4 [18]. This report showed that rSwIL-4 protein is effective in inducing TF-1 human cell proliferation. The rSwIL-4 purified from insect cells stimulated cell growth in a dose-dependent manner and showed considerably higher biological activity than both rSwIL-4 and rHuIL-4 purified from E. coli. The reasons for this observation are currently not understood. This result suggested that the correct protein conformation might be achieved among them. The His-tag at the N-terminus of rSwIL-4 purified from E. coli does not interfere with its receptor binding and signaling as described previously for recombinant canine IL-4 [11]. In addition, the way in which SwIL-4 protein is folded

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in bacteria might be different from the way in which it is folded in insect cells. The possible explanation for this difference may possibly be attributed, at least in part, to differences in glycosylation detectable with tunicamycin tested. The carbohydrates attached to baculovirus-purified rSwIL-4 may have some functions such as the increased stability of the IL-4 molecule in the biological assay or may change the way IL-4 interacts with the IL-4 receptor, either during the initial binding or in subsequent signaling. Thor and Brian [27] reported that the different glycosylation forms of murine IL-4 might have different functional properties on proliferation of the NK cell line in vitro. Further study on the glycosylation of rSwIL-4 will be necessary to clarify the phenomenon. Appropriate glycosylation plays a critical role in the expression and the function of several proteins [28,29,30]. Glycosylation renders a protein more resistance to proteolysis and therefore increases its half-life [31,32]. The glycosylation of cytokines may be of important for the accomplishment of their biological functions, including their biological stability, their interaction with specific receptors and their pharmacokinetic behavior [33]. The role of the post-translational modifications in the biological activity of IL-4 in vivo is left to be determined. However, for expressing physiologically active recombinant proteins such as cytokines, the glycoform of the recombinant protein is considered to be important. The TF-1 cells may be a convenient assay for rSwIL-4, which will aid the optimization of protein expression and purification procedures. The availability of biologically active rSwIL-4 will provide a useful and reproducible tool for further study of this cytokine and its regulatory effect in immune systems in pigs as well as immune responses in swine infectious diseases.

Acknowledgements Authors thank Ms Reiko Satoh, Ryoko Takehara and Yuki Sakuma for technical assistance. Research was funded in part by grant from Ministry of Education, Science, Sports and Culture, and Recombinant Cytokine Project grant number RCP-3210 from the Ministry of Agriculture, Forestry and Fisheries of Japan. This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). Dr Athipoo Nuntaprasert (Faculty of Veterinary Science, Chulalongkorn University, Thailand) is supported by Scholarship from Ministry of Education, Science, Sports and Culture.

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