ISSN 0026-8933, Molecular Biology, 2009, Vol. 43, No. 1, pp. 76–81. © Pleiades Publishing, Inc., 2009. Original Russian Text © H.T. Wu, X.M. Yan, Y.L. Hu, Z.Y. Diao, J. Wu, S.Q. Zhang, 2009, published in Molekulyarnaya Biologiya, 2009, Vol. 43, No. 1, pp. 85–90.
CELL MOLECULAR BIOLOGY UDC 577.152.1
Expression, Characterization of Recombinant Human Soluble Baff Secreted from CHO Cell1 H. T. Wua, b, X. M. Yanc, Y. L. Hua, Z. Y. Diaoa, J. Wuc, and S. Q. Zhanga a
Jiangsu Province Key Laboratory for Molecular & Medical Biotechnology, Nanjing Normal University, Nanjing, 210046 P.R. China e-mail:
[email protected] b Basic Medical College of Nanjing University of Chinese Medicine, Nanjing, 210029 P.R. China c School of Life Science & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240 P.R. China Received November 6, 2007 Accepted for publication February 21, 2008
Abstract—B cell-activating factor of the TNF family (BAFF) is critical for B cell maturation and survival. Here, we constructed a stable CHO cell line, in which the expression level of soluble form of BAFF (sBAFF) was raised from 0.13 µg/ml to 0.55 µg/ml. Purified recombinant sBAFF from these CHO cells not only bound to its receptors but also co-stimulated the proliferation of human peripheral blood B lymphocyte in vitro. These results provided us with a useful basis for further studies about sBAFF-related research. DOI: 10.1134/S0026893309010105 Key words: B cell-activating factor of the TNF family, B lymphocyte, Chinese Hamster Ovary cell. 1
INTRODUCTION B cell-activating factor of the TNF family (BAFF), also known as BLyS, THANK, TALL-1, zTNF4 and TNFSF20, is important in B cell maturation and survival [1]. BAFF is a type II membrane protein that can act in either a membrane-bound form (31.3 kDa) or be proteolytically cleaved into a soluble cytokine (sBAFF, 17 kDa). Its three receptors, B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) and BAFF-receptor (BAFFR), are variably expressed on B cells during their differentiation [2, 3]. The elevated levels of BAFF and its receptors may be involved in the pathogenesis of B cell mediated autoimmune diseases and B-cell malignancy such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and diffuse large cell (DLC) lymphoma [4–6]. Therefore, BAFF, which has agonist or antagonist characteristics, has potential therapeutic applications in B cell-related diseases.
medium was Dulbecco modified essential medium (DMEM, GIBCO BRL), to which 8% (v/v) dialysis fetal bovine serum (GIBCO BRL), 100 U proine/ml, 100 U penicillin/ml, 100 U streptomycin/ml and 50 U kanamycin/ml were added, pH 7. Cells were cultured at 37°C in a water saturated atmosphere with 5% CO2. Construction of secreting plasmid. The human erythropoietion (EPO) signal peptide sequence was fused to sBAFF cDNA. The fused gene was subcloned into plasmids: pcDNA3, pcDNA3.1, pEFneo, respectively. The sBAFF that is biologically active in vitro and in vivo has 125 amino acids truncated from amino acid 134 to amino acid 258 of BAFF (GenBank accession number AF132600). EPO signal peptide-sBAFF fusion cDNA was constructed as follows. First, EPO signal sequence was amplified by PCR with primers: ES1 (5'-GAAC GGTACC ATG GGG GTG CAC GAA TGT CCT GCC TGG CTG TGG CTT CTC CTG TCC CTG C-3') and ES2 (5'- TGG ACC CTG AAC GGC GCC T*AG GAC TGG GAG GCC CAG AGG GAG CGA CAG CAG GGA CAG GA-3'). The 3'-end of primer ES1 and ES2 were complementary to each other. The 5'-end of ES2 was linked with the former 15 bases of sBAFF gene. In addition, EPO signal sequence was mutated (CTG78 to CTA78) in primer ES2 to introduce a Bln I restriction site at the 3' end of signal sequence. Second, sBAFF cDNA was obtained by PCR amplification from the cDNA library of human placenta (Clontech, Palo Alto,
EXPERIMENTAL Bacterial strain, cell line and growth conditions. Escherichia coli TG1 was grown at 37°C in LB medium supplemented with 100 µg ampicillin/ml. CHO-dhfr– cell was cultured with F12 medium (GIBCO BRL) containing 8% (v/v) newborn bovine serum (GIBCO BRL), 100 U penicillin/ml, 100 U streptomycin/ml and 50 U kanamycin/ml, pH 7.2. The selective 1 The
article is published in the original.
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Fig. 1. Schematic diagram of sBAFF expression plasmids. (a) The recombinant gene was subcloned into pcDNA3 plasmid. There was signal sequence from Kpn and Bln sites. pCMV: CMV IE promoter. (b) The recombinant gene was subcloned into pEFneo plasmid. pEF-1-alpha: EF 1-alpha promoter. The amino acid sequence of sBAFF is AVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKVHVFGDELSLVTLFRCIQNMPETLP NNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL.The amino acid sequence of EPO signal peptide is MGVHECPAWLWLLLSLLSLPLGLPVLG.
CA) with primers P1 (5'-GCC GTT CAG GGT CCA G-3') and P2 (5'-G GAATTC TCA CAG CAG TTT CAA TG-3'). Third, using the two DNA PCR products as the templates, we obtained a fused product with primers P3 (5'-GAAC GGTACC ATG GGG GTG CAC-3') and P2 (5'-G GAATTC TCA CAG CAG TTT CAA TG-3') by overlap extension PCR. Finally, this product was ligated into the KpnI/EcoRI sites within pcDNA3 and pcDNA3.1, and KpnI/EcoRV sites within pEFneo (Fig. 1). Cell transfection, stable cell line screen. The plasmids were divided into three groups: plasmid, (1) pcDNA3-sBAFF and pSV-dhfr– (2) pcDNA3–sBAFF and pSV-dhfr plasmid, (3) pEFneo-sBAFF and pSV-dhfr plasmid. Each group, with the molar ratio of 5:1 of the two plasmids, were cotransfected into CHO-dhfr– cells using the calcium phosphate method when cultures were 50–70% confluent. After transfection for 48 h, cells were screened by DMEM selective medium. When dhfr transformants appeared, a clone was isolated, passaged, and then increasingly higher concentration of methotrexate (MTX, 5 × 10–8 mol/l to 5 × 10–7 mol/l) (SigmaAldrich) was added to the cultures to amplify the expression of the transcriptional unit containing the sBAFF gene and dhfr gene. Cell line was considered stable when sBAFF production remained high for more than 15 passages in the absence of MTX selective pressure. Recombinant protein analysis and purification. The supernatant from transfected cells was collected and electrophoresed in 15% SDS-polyacrylamide gel. For western blotting, proteins were transferred to MOLECULAR BIOLOGY
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PVDF membrane (Millipore), and then incubated with rabbit anti-human sBAFF serum (produced in our laboratory) followed by AP-conjugated goat antirabbit IgG (BOSTER, Wuhan, China). The blot was performed using 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitroblue tetrazolium (NBT). The concentration of secreted sBAFF in supernatant was determined by ELISA. The cultural supernatant was collected, centrifuged at 400 g for 10 min to remove cells and debris. ELISA plates were firstly coated with condensed supernatant and then incubated with rabbit anti-human sBAFF serum followed by HRP–goat anti-rabbit IgG (Santa Cruz Biotechnology). The colour development was performed using OPD (o-phenylendiamine, Shanghai Chemicals, China) and measured by a Bio-Rad model 550 microplate reader. By the serial end-point dilution of commercial standard sample (BAFF, Sigma-Aldrich), standard curve was determined by ELISA method. For protein purification, (NH4)2SO4 was added in expression supernatant slowly to a final concentration from 30% to 50% (w/v) saturation. After centrifugation and dialysis, the pellet was dissolved in 50 mmol Tris-HCl (pH 7.2). The sBAFF protein was further purified using DEAE–Sepharose Fast Flow filled column (Amersham–Pharmacia), and eluted with a 10-volume linear gradient from 50 mmol Tris-HCl, 0.1 mol NaCl (pH 7.2) to 50 mmol Tris-HCl, 1 mol NaCl (pH 7.2). The sBAFF containing peaks was collected by Sephacryl S-200 (Amersham–Pharmacia), and eluted with 20 mmol Tris-HCl, 100 mmol NaCl (pH 7.2).
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For N-terminal sequence analysis, the purified protein was transferred to PVDF membrane and subsequently sequenced using Edman degradation with Applied Biosystems Model Procise automated protein sequenator 491 (Applied Biosystems, USA). sBAFF labeling with BNHS. About 20 µl of Biotin space arm-N-hydroxysucoinimide ester (BNHS) (Sigma-Aldrich) that was dissolved in dimethylformamide (DMF) (Amersco, Solon, OH) at a concentration of 1 mg/ml was added to 980 µl of 1 mg/ml sBAFF dissolved in 1 mol/l Na2CO3– NaHCO3, pH 8.5. And these were incubated for 4 h at room temperature, dialyzed overnight against 0.05 mol/l phosphate-buffered saline (pH 7.4). sBAFF and receptors binding assay. Human peripheral blood B lymphocytes were enriched by anti-CD19 magnetic beads (Milenyi Biotech, Germany). Purified cells were > 95% B cells as assessed by expression of CD20. For the flow cytometric analysis, test cells (5 × 105) were washed in phosphatebuffered saline containing 0.5% (v/v) bovine serum albumin (BSA) and incubated with 2 µg/ml BNHSsBAFF or BNHS-BSA control for 30 min at 4°ë. Cells were incubated with PE-streptavidin for 30 min at 4°ë, and analyzed using FACScalibur and CellQuest software (Becton Dickinson). For laser scanning confocal microscopy observation, test cells (5 × 105) were incubated with 2 µg/ml of goat anti-human IgM (Southern Biotechnology Associates) at 37°ë with 5% CO2 for 3 d. After 2 µg/ml BNHS-sBAFF or BNHS-BSA control was added into individual wells for 1 h, the cells were incubated with 10 µl PE-streptavidin at 22°C for 30 min, washed and examined using a Bio-Rad MRC– 1024 with laser scanning confocal microscopy (LSCM). B lymphocyte proliferation assay. For B cell proliferation assay, 5 × 105 B cells suspended in 0.1 ml DMEM medium were placed into individual wells of a 96-well plate, to which 2 µg/ml of goat anti-human IgM was added. After the cells were incubated at 37°C with 5% CO2 for 3 d, the purified sBAFF or commercial standard sample (BAFF, Sigma-Aldrich) was added into individual wells at the final concentration of 2 µg/ml. After 3 d at 37°C, 5% CO2, proliferation was quantitated by MTT method. RESULTS AND DISCUSSION Elevated Expression and Characterization of sBAFF Protein It is important to find a proper system to produce biologically active recombinant sBAFF. Previously, our laboratory has cloned the human sBAFF gene and obtained stable expression in E. coli. However, the prokaryotic expressed sBAFF was present initially in
inclusion bodies and bioactivity was realized only after renaturation. Furthermore, clinical application of bacterially produced products may be affected by the potential presence of endotoxins. Mammalian cells, and in particular the Chinese Hamster Ovary (CHO) cell line has emerged as the preferred host for the large scale production of recombinant proteins for therapeutic use. Recombinant protein from CHO cell has no disadvantages of protein from prokaryotic expression, but also most similar to native protein including molecular structure, physical and chemical characteristic, biology function. In our experiment, cells that were transfected with both pcDNA3-sBAFF and psv-dhfr, or both pcDNA3.1-sBAFF and psv-dhfr, didn’t give rise to dhfr transformant in selective medium. Approximately 35 d after co-transfection, these cells eventually died. On the contrary, approximately 20 d after co-transfection, cells containing both pEFneosBAFF and psv-dhfr plasmids had clones appear in selective medium. Consistently, it was reported that pcDNA3 plasmid was not an appropriate vector for stable expression because it is difficult to integrate into chromosomal DNA in host cells [7]. Moreover, the EF-1α promoter contained in pEFneo plasmid can strongly promote the expression of foreign genes integrated into mammalian chromosomes [8]. Contrary to the CMV IE promoter contained in pcDNA plasmid, the function of EF-1α promoter is irrelevant to the condition of continuous cell division and proliferation [9, 10]. When dhfr transformants appeared in cells co-transfected with pEFneo–sBAFF and psv–dhfr plasmids, a clone was isolated, passaged. Under the increasing concentration of MTX (from 5 × 10–8 mol/l to 5 × 10–7 mol/l), the sBAFF expression level was raised from 0.13 µg/ml to 0.55 µg/ml. MTX, a folate derivative component, blocks DHFR activity completely and irreversibly. Such treatments with stepwise elevated concentrations of MTX may result in the isolation of cells which contain dramatically increased copy numbers of dhfr gene and recombinant gene. Secreted sBAFF protein was detected by SDSPAGE and Western blotting (Fig. 2a), and purified by ion exchange chromatography and size-exclusion chromatography (Fig. 2b, 2c). We sequenced the N-terminal of the purified protein and found that it is identical to the N-terminal sequence of sBAFF (AVQGPEETVT), indicating that the N-terminal EPO signal peptide had been successfully cleaved. The sBAFF Bound to Its Receptors We used flow cytometric analysis to determine the binding of CHO expressed sBAFF to human peripheral blood B cell (Fig. 3a), and observed the binding of sBAFF and its receptors on B cell by LSCM (Fig. 3b). MOLECULAR BIOLOGY
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Fig. 2. The analysis and purification of recombinant protein. (a) SDS-PAGE and Western blotting analyses of sBAFF expressed in CHO cells. M, protein MW marker; lane 1, supernatant from untransfected cells; lane 2, supernatant from transfected cells; lane 3, sBAFF purified from supernatant; lane 4, western blotting for sBAFF expressed from CHO cells. (b, c) Purification of sBAFF expression supernatant. UV spectrum of the purification of sBAFF. After ion exchange chromatography, most of sBAFF was contained in peak 2 and further purified by size-exclusion chromatography. The SDS-PAGE showed that single band, 17 kDa of sBAFF was existed in peak 4.
Fig. 3. The binding of sBAFF receptors on human peripheral blood B lymphocyte. (a) Flow cytometry analysis of sBAFF binding to B cells, HeLa cell as negative control. Cells were stained with BNHS-sBAFF or BNHS-BSA control and than PE-streptavidin. BNHS-sBAFF, in contrast to BNHS-BSA, bound to B cells, but not to HeLa cell. (b) LSCM observation of sBAFF binding B cells. B cells preactivated by anti-IgM were incubated with BNHS-sBAFF and then PE- streptavidin. 1 and 2—B cells were scanned by laser at 565 nm for monolayer. 3−5 and 6—five B cells were scanned flatly 4 times every 2 µm from top to bottom. Each cell showed a cluster binding (indicated by white arrows) besides the entire cell membrane binding on the cell surface. No fluorescence on B cells surface was found in BNHS-BSA control. Scale bar = 10 µm.
Three receptors of BAFF—BCMA, TACI and BAFF-R, are variably expressed on B cell during their differentiation. BAFF-R is the principal receptor for
normal B cell survival and proliferation [11]. Increased expression of BAFF and its receptors has been identified in numerous B-cell malignancies. It is
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ciency (CVID) and panhypogammaglobulinemias [13, 14]. Moreover, serum levels of BAFF are increased in some autoimmune diseases and B-cell malignancy, such as SLE, rheumatoid arthritis, Waldenstrom macroglobulinemia and DLC lymphoma [4, 5, 15, 6]. Accordingly, different BAFF antagonists, anti-BAFF, BAFF-R–Ig and TACI-Ig, are in early clinical trials for human diseases [16].
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Fig. 4. The effect of CHO expressed sBAFF on fresh human peripheral blood B lymphocyte proliferation in vitro. B cells were cultured separately with (1) PBS, (2) anti-IgM alone, (3) sBAFF alone, (4) anti-IgM for 3 d and then commercial standard sample for 3 d, (5) anti-IgM for 3 d and then sBAFF expressed by CHO cell for 3 d, followed by MTT assay. Every group included 6 wells in 96-well plate. Error bars indicated S.D. Under the co-stimulating of anti-IgM and eukaryotic expressed sBAFF (column 5), B cells proliferated over 2 times more than negative controls (column 1, 2 and 3). Meanwhile, the anti-IgM together with commercial standard sample also promoted B cells proliferate to a greater extent (column 4).
possible that altered expression of BAFF receptors may contribute to the progressive accumulation of malignant B cell characteristic [12]. As such, the method in our experiment could be suitable to determine the site and time course of receptor expression and to relate receptor dynamics about sBAFF-mediated function. The sBAFF Promoted Proliferation of B Lymphocyte We found the sBAFF that were produced and secreted into the medium of CHO cells possessed biological activity (Fig. 4). We observed that the activity of the recombinant proteins was most significant at the dose of 2 µg/ml. The sBAFF protein was a anti-IgM co-stimulator of B cell proliferation in vitro. The interaction of anti-IgM with BCR triggered signals is essential for the expression of BAFF receptors on B cell surface. The BAFF may have therapeutic applications in B cell-related disease states such as single IgA, IgG, and IgM deficiencies, common variable immunodefi-
In our experiment, the CHO expression system can be used to produce fully functional sBAFF protein, which showed some different qualities in contrast with prokaryotic expressed sBAFF, such as better stability and weak antigenicity. Now we obtained its antibody by the help of recombinant sBAFF protein. The antibody was used not only in BAFF antagonist research, but also to develop an ELISA kit for detection of BAFF in human serum. ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of Jiangsu Province (no. BK2005140) and Specialized Research Fund for the Doctoral Program of Higher Education. REFERENCES 1. Schneider P., MacKay F., Steiner V., Hofmann K., Bodmer J.L., Holler N., Ambrose C., Lawton P., Bixler S., Acha-Orbea H., Valmori D., Romero P., Werner-Favre C., Zubler R.H., Browning J.L., Jürg Tschopp J. 1999. BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J. Exp. Med. 189, 1747–1756. 2. Gross J.A., Johnston J., Mudri S., Enselman R., Dillon S.R., Madden K., Xu W., Parrish-Novak J., Foster D., LoftonDay C., Moore M., Littau A., Grossman A., Haugen H., Foley K., Blumberg H., Harrison K., Kindsvogel W., Clegg C.H. 2000. TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease. Nature. 404, 995–999. 3. Thompson J.S., Bixler S.A., Qian F., Vora K., Scott M.L., Cachero T.G., Hession C., Schneider P., Sizing I.D., Mullen C., Strauch K., Zafari M., Benjamin C.D., Tschopp J., Browning J.L., Ambrose C. 2001. BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF. Science. 293, 2108–2111. 4. Zhang J., Roschke V., Baker K.P., Wang Z., Alarcön G.S., Fessler B.J., Bastian H., Kimberly R.P., Zhou T. 2001. Cutting edge: A role for B lymphocyte stimulator in systemic lupus erythematosus. J. Immunol. 166, 6–10. 5. Cheema G.S., Roschke V., Hilbert D.M., Stohl W. 2001. Elevated serum B lymphocyte stimulator levels in patients with systemic immune-based rheumatic diseases. Arthritis Rheum. 44, 1313–1319. 6. Novak A.J., Grote D.M., Stenson M., Ziesmer S.C., Witzig T.E., Habermann T.M., Harder B., Ristow K.M., Bram R.J., Jelinek D.F., Gross J.A., Ansell S.M. 2004. Expression of BLyS and its receptors in B-cell nonMOLECULAR BIOLOGY
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