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Veterinary Immunology and Immunopathology 128 (2009) 374–380 Contents lists available at ScienceDirect Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm Research paper Molecular cloning, expression, and bioactivity of dove B lymphocyte stimulator (doBAFF) Wuguang Lu a,b, Peng Cao b,*, Xueting Cai b, Jiemiao Yu b, Chunping Hu b, Meng Cao b, Shuangquan Zhang a,* a b Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, 210097, Jiangsu, China Laboratory of Cellular and Molecular Biology, Jiangsu Province Institute of Traditional Chinese Medicine, Nanjing, 210028, Jiangsu, China A R T I C L E I N F O A B S T R A C T Article history: Received 25 July 2008 Accepted 19 November 2008 B cell activating factor (BAFF) belonging to the tumor necrosis factor (TNF) family is a novel member of the tumor necrosis factor ligand family and plays an important role in B lymphocyte maturation and survival. cDNA of dove B lymphocyte stimulator (doBAFF) was amplified from total RNA of dove spleen by RT-PCR (reverse transcription PCR). The open reading frame of doBAFF consists 867 bases encoding a protein of 288 amino acids. Sequence comparison indicated the amino acid sequence of doBAFF showed high identity to hBAFF (50.66%) and cBAFF (91.32%). The result of RT-PCR showed that doBAFF was highly expressed in the spleen and bursa of fabricius. To enhance the soluble expression of doBAFF in Escherichia coli, we fused the extracellular region of doBAFF gene with a small ubiquitin-related modifier gene (SUMO) by over-lap PCR. The resulting fused protein SUMO-sdoBAFF was highly expressed in DE3(BL21) with a molecular weight of 35 kDa. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease, Ulp1. The sdoBAFF protein was further purified by Ni-NTA affinity chromatography. In vitro, the MTT assays indicated that the purified doBAFF as well as SUMO-sdoBAFF proteins were able to promote bursa lymphocyte survival in dosedependent manner. ß 2008 Elsevier B.V. All rights reserved. Keywords: Dove B lymphocyte stimulator SUMO Fusion protein B cells survival 1. Introduction Members of the tumor necrosis factor (TNF) family and their receptors are important regulators of the immune system (Mak and Yeh, 2002; Gordon et al., 2003; Kern et al., 2004; Kalled et al., 2005; So et al., 2006; Pelekanou et al., 2008). The tumor necrosis factor family member, BAFF also known as BlyS, TALL-1, THANK, zTNF4 and TNFSF13B, plays an important role in B lymphocyte maturation and survival (Batten et al., 2000; Gao et al., 2007). BAFF is a type II membrane * Corresponding author. Tel.: +86 25 85608666; fax: +86 25 85608666. E-mail addresses: [email protected] (P. Cao), [email protected] (S. Zhang). 0165-2427/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2008.11.026 protein that can function as the membrane-bound form or be proteolytically cleaved into a soluble cytokine (sBAFF) by a protease of the furin family (Schneider et al., 1999; Bodmer et al., 2002). In vitro, soluble BAFF was found to promote B cell survival and co-stimulate B cell proliferation with anti-IgM (Schneider et al., 1999; Moore et al., 1999). Overexpression of BAFF is closely involved in the pathogenesis and progression of many kinds of autoimmune disorders (De Vita et al., 2008). Therefore, BAFF has been considered as an ideal therapeutic target for these conditions. Most of the previous researches mainly focus on mammalian BAFF, such as human BAFF and mouse BAFF drive the differentiation of immature B cells into more mature cells and also to promote B cell survival and antibody production (Khare et al., 2000; MacLennan and W. Lu et al. / Veterinary Immunology and Immunopathology 128 (2009) 374–380 Vinuesa, 2002; Sakurai et al., 2007; Gilbert et al., 2006). Recently, the bioactivity of BAFF in the avian species was taken close attention. Chicken BAFF (cBAFF) has been proved that can promote the growth and maturation of follicular B cells in bursa of fabricius (Koskela et al., 2004; Guan et al., 2007). Furthermore, recombinant soluble duck BAFF (duBAFF) and goose BAFF (gsBAFF) purified from Escherichia coli have a positive effect on bursa B cells survival and proliferation (Guan et al., 2007; Dan et al., 2007). Especially, because of high conservation of BAFF in the evolution, functional cross-reactivity exists between mammalian and avian BAFF (Guan et al., 2007). In this paper, we at the first time report the molecular cloning and bioactivity analysis of doBAFF. 375 Table 1 Primers used in this research. Primers Sequence (50 –30 ) doBAFF-1 doBAFF-2 ATGAAATCCGTGGACTGTGTGCACGTC ACTGAGCCCGGGTCAGAAGAGTCTGACGGCACCACGATAT sdoBAFF-F sdoBAFF-R GGA AT TCCATATGTCTGTTGTCCACAC CGGGATCCT CAGAAGAGTCTGACGGCAC b-Actin-F b-Actin-R TGATATTGCTGCGCTCGTTG TCATTGTA GAAAGTGTG SUBAFF-F SUBAFF-R GGAG GTTCTG TTGTCC ACAC CGGGATCCTCAGAAGAGTCTGACGGCAC SUMO-F CGATATACCATGGGTCATCACCATCATCATCACG GGTCGGACTCAG GTGTGGACAACAGAACCTCCAATCTGTTCGCGGTG SUMO-R 2. Materials and methods 2.1. Animals and cell preparations 10-week-old doves (Columba livia domestica) were purchased from Yuanqiao Meat pigeon farms of Rugao city of Jiangsu, China. The dove lymphocyte was separated from bursa of fabricius homogenate using lymphocyte isolation solution (TBD sciences, China) according to the manual. All cells were maintained in RPMI 1640 medium with penicillin/streptomycin (Gibco BRL, US) supplemented with 10% FCS at 37 8C in a CO2 incubator. 2.2. RNA extraction, cDNA synthesis and sequence comparison Tissues of dove such as heart, spleen, kidney, liver and bursa of fabricius were collected and stored in 80 8C ultra cold freezer after dissect. The total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, US) according to the standard protocol. RNA quality was assessed by electrophoresis on 1% agarose gel. Total RNA was treated with RQ1 RNase-free DNase (Promega, US) to remove contaminated DNA. To synthesize cDNA by reverse transcription, 1 mg total RNA, 10 units Reverse Transcriptase XL (AMV) (Takara, Japan) and 50 pmol Random primer (6 mer) were reacted for 1 h at 42 8C in 20 ml mixture according to the manufacturer’s instruction. Degenerate primers doBAFF-1 (Table 1) and doBAFF-2 (Table 1) were used in the PCR. Their design was based on regions of high homology among the sequences of duck, human and mouse. PCR was performed by the following procedure: denature for 30 s at 94 8C, annealing for 30 s at 54 8C, and extension for 1 min at 72 8C, and then 72 8C for 8 min. PCR products from a 1% agarose gel were purified with the gel purification kit and subcloned into PMD 18-T vector (Takara, Japan). Positive recombinant colony was characterized by PCR and further confirmed by DNA sequencing. The amino acid sequence of doBAFF protein was compared with avian and mammalian BAFFs from the GenBank using DNAMAN 10 software. The phylogenetic analysis was conducted using neighbor joining (NJ) and minimum evolution, based on amino acid sequences. The sequence data were transformed into a distance matrix (pdistance). The NJ tree was obtained using MEGA 4.0 with complete deletions of gaps, and was validated with the minimum evolution tree. One thousand bootstraps were performed for the NJ trees to verify results reliability. 2.3. RT-PCR analysis of doBAFF mRNA expression in tissues The expression of doBAFF was investigated using RTPCR. Equivalent amounts of total RNA, isolated from bursa of fabricius, spleen, kidney, heart and liver were reverse transcribed into cDNA as the template for PCR, first-strand cDNA was obtained from total RNA using PrimeScriptTM RT-PCR Kit (Takara, Japan) according to the standard protocol. A variable amount of the cDNA was used in a total volume of 50 ml of a PCR mixture using Premix Ex TaqTM. PCR amplification of doBAFF cDNA was carried out for 30 cycles, with primers sdoBAFF-F (Table 1) and sdoBAFF-R (Table 1). Amplification was performed at 94 8C for 5 min and 94 8C for 30 s, 50 8C for 30 s, 72 8C for 45 s, finally amplificated 6 min at 72 8C. PCR products were run on a 1% agarose gels. Columba livia b-actin (GenBank accession number: DQ022673) expression was used as an internal control for RNA content and integrity. 2.4. Construction of SUMO-sdoBAFF fusion expression vectors The DNA fragment encoding the extracellular region of doBAFF (aa137–288) also known as the soluble form of doBAFF (sdoBAFF) was amplified by PCR using primers SUBAFF-F and SUBAFF-R (Table 1) to produce the fusion gene encoding SUMO-sdoBAFF, the (small ubiquitin-like modifier) SUMO fragment was amplified from a baculovirus expression plasmid using primers SUMO-F and SUMO-R (Table 1). The SUMO-R and SUBAFF-F had the overlapping complementary sequence, while SUMO-F and sdoBAFF-R had NcoI with His6 tag and BamHI recognition sites for directed cloning into vector. After the first round of PCR using SUMO-F/SUMO-R and SUBAFF-F/sdoBAFF-R, gel purification was performed for the two products. A second round of PCR followed using the two resulting PCR products as well as the SUMO-F and sdoBAFF-R primers to obtain the fragment encoding SUMO-sdoBAFF. Then the fragment was digested with NcoI and BamHI and inserted in a frame into pET28a plasmid (Novagen, US). DH5a competent 376 W. Lu et al. / Veterinary Immunology and Immunopathology 128 (2009) 374–380 cells were first transformed and screened in Luria– Bertani (LB) medium containing kanamycin (50 mg/ml). The plasmids were then confirmed by endonuclease restriction digestion assay and further confirmed by DNA sequencing. The resulting plasmid was named pET28a/ SUMO-sdoBAFF. The construction of pET28a/His-sdoBAFF was performed according to Cao et al. (2005) with primers sdoBAFF-F and sdoBAFF-R which had NdeI and BamHI recognition sites for directed cloning into vector. 2.5. Expression and purification of SUMO-sdoBAFF protein Inoculate a 100 ml culture (LB, 50 mg/ml kanamycin) 1:50 with the noninduced overnight bacterium and grow at 37 8C with vigorous shaking until an OD600 of 0.6 is reached. Then induce expression by adding 0.4 mM IPTG and cultivate for additional 48 h at 15 8C, 150 rpm. The soluble protein in the supernatant was collected by refrigerate centrifugation after hypersound quassation. Finally, the target protein was purified with His-Bind Columns (Qiagen, Germany) according to the manual. 2.6. SDS-PAGE and Western blotting Protein was separated on a 13% polyacrylamide gel under reducing conditions and then transferred to a PVDF membrane (Malakhov et al., 2004). The membrane was soaked for 15 min with transfer buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH 8.3) and nonspecific protein binding was blocked by incubating the Fig. 1. The open reading frame (ORF) of doBAFF cDNA sequence (GenBank accession no. EU334145). The predict transmembrane region is underlined and the furin cleavage site is shown in italics. W. Lu et al. / Veterinary Immunology and Immunopathology 128 (2009) 374–380 membrane with 5% MPBS (5% skim milk in PBS, pH 7.4) for 1 h. After washing with PBST (PBS containing 0.05% Tween 20) 15 min, 1:1000 anti-His6 IgG mAb (Novagen, US) was added into MPBS and incubated for 2 h at 37 8C. The membrane was washed three times for 10 min with PBST, incubated for 60 min with a 1:5000 dilution of goat antimouse IgG conjugated with horseradish peroxidase (HRP) (Santa Cruz Biotechnology, US) in PBST, and subsequently washed three times for 5 min with PBST. The blot was performed using the tetramethyl benzidine (TMB) chemiluminescence system (Promega, US). 377 2.7. SUMO fusion enzymolysis N-terminal His-tagged SUMO protease 1, Ulp1 was prepared in our laboratory. Mix the purified Ulp1 and SUMO-sdoBAFF at a proportion of 1:20, dialyze the purified SUMO-sdoBAFF and Ulp1 mixture at least 24 h at 4 8C reaction buffer (50 mM Tris–HCl, 150 mM NaCl, 0.5 mM EDTA, pH 7.9, 10% glycerol). During the dialysis, change the buffer (1 L) two times to effectively remove the detergent and imidazole. The digest was purified using Ni-NTA resin (Qiagen, Germany). SUMO with N-terminal His-tag was Fig. 2. Comparison of protein sequences from dove, goose, chicken, duck, human and mouse. Sequence of doBAFF (GenBank accession no. EU334145), gsBAFF (GenBank accession no. DQ874394), dBAFF (GenBank accession no. DQ445092), cBAFF (GenBank accession no. AF506010), mBAFF (GenBank accession no. AF119383), hBAFF (GenBank accession no. AF116456) were aligned using the DNAMAN program. Grey shading indicates identical residues among the six sequences. 378 W. Lu et al. / Veterinary Immunology and Immunopathology 128 (2009) 374–380 bound on the column and sdoBAFF was washed out with 500 mM NaCl 20 mM Tris–HCl 50 mM imidazole. Then the purified sdoveBAFF was dialyzed against TEG (50 mM Tris– HCl pH 7.9, 50 mM NaCl, 0.5 mM EDTA 5% glycerol) at least 12 h. 2.8. Lymphocytes of fabricius, bursa proliferation assay MTT assay was used to evaluate the number of viable cells in each group at different concentrations. For lymphocyte cell survival assay, the isolated lymphocyte cells were adjusted to 105 cells/ml and cultured in RPMI 1640 supplemented with 10% FCS and 100 U/ml penicillin/ streptomycin (Gibco BRL, US) in triplicate in 96-well flatbottomed plates. After 4 h accommodative cultivate, different concentrations SUMO-sdoBAFF and sdoBAFF were added to stimulate the cells. As controls, stimulated with His-SUMO in the same concentration gradient. The cells were incubated at 37 8C in a humidified atmosphere with 5% CO2 for 72 h. Cell survival was measured by the MTT method with multiskan spectrum reader (Thermo, US). Fig. 3. Phylogenetic tree of BAFFs between dove, goose, chicken, duck, human and mouse. The tree was generated by MEGA 4.0 software using the neighbor-joining method. Lengths of horizontal lines indicate genetic distance. The bootstrap values from 1000 replications are indicated at the inner nodes. 3. Results 3.1. The cDNA cloning of doBAFF The cDNA of doBAFF encoding 288 amino acid residues was cloned from liver tissue by RT-PCR. As shown in Fig. 1, structural analysis indicated that the doBAFF protein has a predict transmembrane spanning domain and a putative furin protease cleavage site similar to other avain and mammalian BAFFs. 3.2. Sequence comparison As shown in Fig. 2, sequence comparison indicated that the amino acid sequence of doBAFF shared high identity to hBAFF (50.66%), mBAFF (44.69%), dBAFF (90.63%), cBAFF (91.32%), gsBAFF (91.67%), respectively. Phylogenetic analysis is based on amino acids sequence information. Phylogenetic analysis of these species was performed at the amino acid level. The phylogenetic tree of the BAFF proteins revealed that these BAFFs were divided into two Fig. 4. Expression of doBAFF in various tissues were analyzed by RT-PCR. b-Actin was used as a internal control for the amount and quality of mRNA. The position control RNA was provided by the RT-PCR kit. groups: doBAFF, cBAFF, dBAFF, gsBAFF and hBAFF, mBAFF. As shown in Fig. 3, all of the BAFFs originated from only one root. DoBAFF, cBAFF, dBAFF, gsBAFF constituted one subgroup. This same pattern of separation was maintained when the minimum evolution analysis was used. 3.3. Tissue-specific expression of doBAFF mRNA in various tissues was analyzed by RT-PCR. As shown in Fig. 4, the doBAFF mRNA were extensively expressed in various tissues, whereas high levels of expression of the doBAFF was observed in the spleen and bursa of fabricius. In addition, expression was weakly detected in the heart, liver, and kidney. This suggested that Fig. 5. (A) Expression analysis of His-doBAFF in BL21(DE3): lane 1, bacterium without IPTG introduction; lane 2, bacterium introduced with 0.4 mM IPTG in 15 8C; lane 3, the supernatant after hypersound quassation and hypothermy centrifugalization. (B) Expression and purification analysis of SUMO-sdoBAFF: lane 1, bacterium without IPTG introduction; lane 2, bacterium introduced with 0.4 mM IPTG in 15 8C; lanes 3–7, purified SUMO-sdoBAFF with nickelaffinity chromatography. (C) Western blot analysis of SUMO-sdoBAFF with mAb against His6 tag. W. Lu et al. / Veterinary Immunology and Immunopathology 128 (2009) 374–380 379 doBAFF was the predominant form expressed in lymphatic organ. As controls, b-actin generated specific bands of similar intensity in nearly all tissues. 3.4. Soluble expression of sdoBAFF protein by SUMO-fusion As shown in Fig. 5A, When fused with His6 tag without SUMO, soluble sdoBAFF was poorly expressed in E. coli and could not be detected in the supernatant. On the contrary, a relative concentration of SUMO-sdoBAFF fusion protein was detected by SDS-PAGE further by Western blot (Fig. 5B and C). Must be point out that SUMO is about 10 kDa but runs on an SDS-PAGE gel as 20 kDa. As a result, SUMOsdoBAFF runs as 35 kDa, even though SUMO-sdoveBAFF is actually 25 kDa. 3.5. Enzymolysis of SUMO-sdoveBAFF with Ulp1 As shown in Fig. 6, after diluted and cleaved by SUMOspecific protease Ulp1, the fusion protein release a 20 kDa fragment SUMO dimmer and soluble doBAFF about 15 kDa. After flow through the His-Bind Columns, nearly all the undigested SUMO-sdoBAFF and SUMO with N-terminal His-tag were bound on the column. Fig. 7. Effects of different concentrations sdoBAFF and SUMO-sdoBAFF to the survival of lymphomonocyte of dove bursa of fabricius in vitro. Fresh isolated lymphomonocyte of dove bursa of fabricius were incubated with sdoBAFF and SUMO-sdoBAFF at varying concentrations for 72 h. lymphomonocyte of dove bursa of fabricius, freshly isolated bursa lymphocytes were cultured with sdoBAFF protein. As shown in Fig. 7, after 72 h, although cell death occurred rapidly due to apoptosis, sdoBAFF dramatically prolonged the survival of bursa B cells in culture, and represented a dose-dependent manner to sdoBAFF treatment. The control protein SUMO did not have any survival effect. 4. Discussion 3.6. Bioactivity of recombinant sdoveBAFF and SUMOsdoBAFF Both SUMO-sdoBAFF and sdoBAFF could promote the survival of lymphocyte of dove bursa of fabricius in vitro. To test the effects of sdoveBAFF to the survival of Fig. 6. SDS-PAGE analysis of SUMO-sdoBAFF digested by SUMO protease and purified sdoBAFF. Lane 1, purified fusion SUMO-sdoBAFF using HisBind Columns; lane 2, digestion of purified fusion SUMO-sdoBAFF using SUMO protease at 4 8C for 24 h; lane 3, the purified sdoBAFF obtained after reloading the digested sample thought the Ni-NTA column; lane 4, protein marker. In the present study, the cDNA sequence of B cell activating factor from Columba livia domestica was determined. The cloning strategy was based on nucleotide sequence homology between the dove and the other poultry. The protein of doBAFF containing a predicted transmembrane domain and a putative furin protease cleavage site like cBAFF, hBAFF and mBAFF. Amino acids sequence comparison revealed that the doBAFF has a very high sequence and structural similarity to other avian BAFFs. From the phylogenetic analysis of BAFF, we detected the genetic relationship between dove, chicken, duck, mouse and human. The cladogram indicated that doBAFF showed a high homology with other BAFFs especially the avian BAFFs. DoBAFF mRNA was extensively expressed in various tissues, especially in the lymphatic organs such as spleen and bursa of fabricius which were rich with B achroacyte. As the same as many other heterogenesis proteins, recombinant BAFF expression in E. coli remains difficult. Refolding technology and traditional fusion technology were used to enhance the soluble expression (Guan et al., 2007; Cao et al., 2005). But low yield, low reproducibility of refolding technology and inefficient cleavage of traditional fusion systems were major problems we have faced. SUMO both enhanced expression and cleavage of the fusion protein. The effect that SUMO has on enhancing protein solubility can be explained in part by the structure of SUMO. SUMO has an external hydrophilic surface and inner hydrophobic core, which may exert a detergent-like effect on otherwise insoluble proteins (Butt et al., 2005). The SUMO-specific protease Ulp1 cleave the conserved Gly–Gly motif at the C-termini of SUMO from recombinant fusion protein efficiently in suitable conditions. Unlike EK or TEV protease, whose recognition sequences are short and degenerate, Ulp1 recognizes the tertiary sequence of 380 W. Lu et al. / Veterinary Immunology and Immunopathology 128 (2009) 374–380 SUMO. As a result, Ulp1 never cleaves within the fused protein of interest so we can get intact protein of interest with little exogenous amino acid residue. We cloned the sdoBAFF at the 30 end of the His6-SUMO gene by over-lap PCR. However, when introduced in 37 8C, both SUMOsdoBAFF and His-sdoBAFF were most inclusion body (data was not shown). So hypothermy induction was used to improve the yield of soluble proteins. Western blot analysis of SUMO-sdoBAFF purified from E. coli revealed a prominent band at 35 kDa, indicative of SUMO-sdoBAFF soluble expression while nearly no His-sdoBAFF was detected in the supernatant of lysate even though in the same introduce condition. BAFF is a potent survival factor for B cells in mammals, chicken, goose and duck, while the soluble forms of hBAFF and mBAFF were biologically active in promoting survival of B cells treated with anti-IgM in vitro. This also holds true for the dove system. MTT assays indicated that both sdoBAFF and SUMO-sdoBAFF can promote the survival of bursa lymphocyte in dose-dependent. In summary, this work determined the cDNA sequence of doBAFF, and analyzed doBAFF mRNA expression in tissues. Moreover, the construction of SUMO expressing system supplied an universal method for high level expression soluble proteins. 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