Expression and purification of buffalo interferon-tau and efficacy of recombinant buffalo interferon-tau for in vitro embryo development

Cytokine xxx (2015) xxx–xxx

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Expression and purification of buffalo interferon-tau and efficacy of recombinant buffalo interferon-tau for in vitro embryo development Shrabani Saugandhika a, Vishal Sharma a, Hrudananda Malik a, Sikander Saini a, Sudam Bag a, Sudarshan Kumar a, Niraj Kumar Singh b, Ashok Kumar Mohanty a, Dhruba Malakar a,⇑ a b

Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana 132001, India Animal Biotechnology Centre, GADVASU, Ludhiana, Punjab, India

a r t i c l e

i n f o

Article history: Received 7 January 2015 Received in revised form 4 March 2015 Accepted 12 March 2015 Available online xxxx Keywords: Buffalo Isoform Interferon tau Blastocyst Trophectodermal cells

a b s t r a c t The aim of our study was to optimize growth and induction parameters, for expression and large scale purification of functionally active buffalo interferon tau, and to study its possible impact on in vitro blastocyst development. The buffalo interferon-tau gene (BuIFN-T1) bearing gene bank accession No. JX481984, with signal sequence, was obtained through polymerase chain reaction (PCR) from bovine early embryos and was cloned into pJET vector. After being verified, the fragments without signal sequence, were inserted into the expression vector pET-22b and the recombinant plasmid was induced to express the recombinant protein in a prokaryotic expression system. The recombinant BuIFN-T was confirmed by SDS–PAGE and Western blot and subjected to three steps of large scale purification using His Affinity chromatography, Anion Exchange chromatography and Gel Filtration chromatography. The purified recombinant BuIFN-T protein was validated by mass spectroscopy analysis. To examine the effect of recombinant BuIFN-T protein on developmental competency of buffalo embryos, purified recombinant BuIFN-T protein was added to in vitro embryo culture medium (at concentration of 0, 1 lg/ml, 2 lg/ml, 4 lg/ml) for 9 days. Addition of recombinant BuIFN-T (2 lg/ml) significantly improved the rate of blastocyst production, 45.55% against 31.1% control (p < 0.01). Here we conclude that the recombinant BuIFN-T was successfully purified to homogeneity from a prokaryotic expression system and it significantly increased the blastocyst production rate in buffalo. These findings suggest a potential impact of IFN-T in promoting embryonic growth and development. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Interferon-T (IFN-T) is a subclass of type I IFNs, described as a family of protein hormones produced by the trophoblast of conceptuses of ruminant ungulates that signals for maternal recognition of pregnancy [1,2]. The preimplantation conceptus secretes IFN-T into the uterine lumen during early pregnancy to alter maternal physiology and allow establishment of pregnancy [1,2]. The maternal recognition of pregnancy (MRP) and subsequently

Abbreviations: IFN-T, interferon-T; BuIFN-T, buffalo interferon-tau gene; MRP, maternal recognition of pregnancy; PGF2, prostaglandin F2 alpha; PGE, prostaglandin E; GM-CSF, granulocyte macrophage colony stimulating factor; UCRP, ubiquitin cross reactive protein; rBuIFN-T, recombinant BuIFN-T; IMAC, Immobilized-metal affinity chromatography; QSCC, Q sepharose column chromatography; IEX, ion exchange chromatography; GEC, gel exclusion chromatography; m.o.i, multiplicity of infection; CPE, cytopathic effect; JE, Japanese encephalities. ⇑ Corresponding author. Tel.: +91 9416741839. E-mail address: [email protected] (D. Malakar).

embryonic implantation both are mediated by antiluteolytic, immunogenic and luteotrophic functions of IFN-T [3–5]. IFN-T acts on uterine epithelium, suppresses transcription of the genes for estrogen receptor and oxytocin receptor and blocks release of prostaglandin F2 (PGF2) alpha and thus prevents luteolysis and maintains pregnancy [6–8]. IFN-T upregulates the genes required for Prostaglandin E (PGE) synthesis and brings a shift in PGs to PGE2 over PGF2 alpha’ which induces Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) that modulates local immune functions at the fetomaternal interface and stimulates the growth of conceptus [9,10]. IFN-T also upregulates several other endometrial proteins (e.g. Ubiquitin cross reactive protein (UCRP), 20 ,50 oligoadenylate synthetase, Mx protein, etc.) during early pregnancy, which play a role in the establishment of pregnancy and growth of conceptus [11–13]. IFN-T is produced by mononuclear trophoblast cells of ovine, bovine, and caprine conceptuses and has been detected using molecular probes in all ruminant ungulates studied [14,15].

http://dx.doi.org/10.1016/j.cyto.2015.03.012 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Saugandhika S et al. Expression and purification of buffalo interferon-tau and efficacy of recombinant buffalo interferontau for in vitro embryo development. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.03.012

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In addition to its role in pregnancy, IFN-T also possesses potent antiviral, antiproliferative, and immunomodulatory activities similar to other type I IFNs. Interestingly, IFN-T lacks the cytotoxicity associated with IFN-A [16–18]. This has focused attention on IFN-T as a potential therapeutic agent to treat cancer and viral diseases [2,19]. For example, ovine IFN-T and human interferon alpha (IFN-A) were equally effective in blocking human and feline immunodeficiency virus replication in human and feline lymphocytes, respectively [20]. In this system, human IFN-A was toxic to cells at greater than 1000 antiviral units/ml, whereas OvIFN-T was not toxic at doses up to 500,000 antiviral units/ml [21]. Because of the potential of IFN-T as a fertilityregulating agent in ruminants and as a therapeutic to treat diseases in humans and animals, a large amount of protein is required for experimentation. Several reports have been published on the heterologous expression of bovine and ovine IFN-T where most of the studies reports use of eukaryotic expression system for production of recombinant IFN-T [22–25]. These studies described successful purification of ovine and bovine IFN-T expressed in Pichia pastoris and silk worm, respectively [23–25]. There are also a few studies reporting expression of bovine IFN-T in prokaryotic expression system but expression of recombinant bovine IFN-T is reported in terms of GST tagged fusion protein or in the form of inclusion bodies where further techniques have been employed to derive pure recombinant IFN-T protein from fusion tags and inclusion bodies [26–28]. Thus, the present study was initiated with an idea to produce recombinant interferon tau in functional form (biologically active form) in Escherichia coli which would be a cheaper host for recombinant protein production and to standardize optimal purification parameters to get yield comparable to methods adopted by others [23–28]. Moreover as far as buffalo species is concerned, to our information it is for the first time we report production of biologically active recombinant buffalo IFN-T using a prokaryotic expression system. The role of IFN-T, in ruminants, has been confirmed in maternal recognition of pregnancy and subsequent embryonic implantation [29]. It is known that IFN-T exerts its biological activities by binding to the type I interferon receptor (IFNAR), which consists of 2 transmembrane chains, IFNAR1 and IFNAR2 [30]. A recent report demonstrated the expression of IFNAR1 receptor in bovine embryos from morulae to blastocysts stage during in vitro embryonic development [31]. Furthermore, it is reported that the culture medium when supplemented with IFN-T remarkably promoted the proliferation of ovine trophectoderm and in vitro development of bovine embryos [31,32]. Therefore it is possible that IFN-T may play some role in early embryonic development. At present there is no direct evidence that IFN-T has effect on embryonic development in buffalo embryos. In this study to investigate the effects of IFN-T on the early embryonic development in buffalo, the conditions for large scale expression and purification of the buffalo IFN-T in prokaryotic expression system were optimized. To confirm the hypothesis that IFN-T plays a role in vitro embryonic development we examined the effects of recombinant IFN-T on in vitro development of blastocysts in buffalo. Furthermore we also studied the relative expression of IFN-T in preimplantation embryos.

2.2. Cloning and expression of buffalo interferon tau (BuIFN-T) 2.2.1. Cloning of buffalo interferon tau (BuIFN-T) In vitro development of Buffalo blastocysts was carried out following the protocol as mentioned by [33]. RNA was isolated from in vitro developed buffalo blastocysts using the RNeasy mini kit (Qiagen Corp., Carlsbad, CA) according to the manufacturer’s instructions. The forward primer (50 -AACCTACCTGAAGGTTCACC CAGA-30 ) and reverse primer (50 -TGAGTGTACGAAGGTGATGTG GCA-30 ) were designed based on the published sequence of BuIFN-T1 gene from GenBank accession No. JX481984. For amplification of BuIFN-T, two micrograms of tcRNA was incubated at 65 °C for 5 min, then reverse transcribed with M-MuLV reverse transcriptase (#K1621, Fermentas Corp, USA), oligo (dT) primer, and 10 mM each of dNTP mix at 42 °C for 60 min. PCR amplification of IFN-s genes were performed with high fidelity dream taq DNA Polymerase (Fermentas Corp, USA) and bovine IFN-s specific primers in a total reaction volume of 20 ll. Briefly, 10 ll of dream taq™ green PCR master mix (Fermentas Corp, USA), 8 ll of NFW, 0.5 ll of each primer and 1 ll of cDNA. The cyclic conditions used for PCR amplification were as: initial denaturation at 94 °C for 3 min; 35 cycles of 94 °C for 30 s; 62 °C for 30 s and 72 °C for 1 min; and final extension at 72 °C for 10 min. PCR product was purified on 1.5% agarose and ligated into the pJET1.2/blunt cloning vector (Fermentas Corp. USA) and used to transform TOP10 competent E. coli cells according to the manufacturer’s instructions (Invitrogen Inc., Carlsbad, CA, USA). 2.2.2. Construction of prokaryotic expression vector of BuIFN-T TOP 10 cells harboring BuIFN-T1 gene in the pJET cloning vector was verified by sequencing, BuIFN-T gene without signal sequence were generated from p-JET cloning vector using 50 -end primers with NdeI restriction sites (50 -ATCGCATATGTGTTACCTATCTC GGAGACTCATG-30 ) and 30 end primer with XhoI restriction site (50 -ATCGCTCGAG AGGTGAGTTCAGATTTCCACCCAT-30 ). The cyclic conditions used for PCR amplification were as: initial denaturation at 94 °C for 3 min; 35 cycles of 94 °C for 30 s; 62 °C for 30 s and 72 °C for 1 min; and final extension at 72 °C for 10 min using dream taq™ green PCR master mix and Taq polymerase (Fermentas Corp, USA). Amplified products were cloned into pET22b vector after double restriction digestion and were used to transform competent BL21(DE3) E. coli cells. 2.2.3. Expression of recombinant BuIFN-T The ampicillin resistant colonies containing recombinant plasmid pET-22b-BuIFN-T were grown to an OD 0.6 at 600 nm and induced with 1 mM, 0.5 mM or 0.1 mM IPTG. Both the cell lysates and cell pellets were analyzed for expression analysis of all the recombinant constructs on SDS–PAGE. To optimize the expression of BuIFN-T in soluble fraction, recombinant clones were induced at three different temperatures; 37 °C, 16 °C and 12 °C. Soluble protein from respective cell lysates was extracted using Q proteome Bacterial Protein prep kit (Qiagen, USA) according to manufacturer’s instruction. The soluble and insoluble fractions of proteins from the pellet were subjected to SDS–PAGE and western blot analysis. 2.3. Purification of recombinant BuIFN-T (rBuIFN-T)

2. Materials and methods 2.1. Ethics statement No ethical approval was needed for oocyte collection because the ovaries were obtained from the Local Slaughter House, Karnal, India and the animals were not killed for scientific research.

2.3.1. Affinity purification using immobilized-metal affinity chromatography (IMAC) column For purification of soluble rBuIFN-T, BL21(DE3) cells harboring pET22b-BuIFN-T expression constructs was cultured in 2 l volume of LB broth for 22 h at 12 °C with 50 lg/ml ampicillin. Cells were harvested by centrifugation at 6000 g for 20 min and resuspended in His binding buffer (0.3 M NaCl, 10 mM imidazole in 50 mM

Please cite this article in press as: Saugandhika S et al. Expression and purification of buffalo interferon-tau and efficacy of recombinant buffalo interferontau for in vitro embryo development. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.03.012

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phosphate buffer) and subjected to sonication (conditions – 40 amplitude, 5 s pulse, 5 s on/off for 30 min duration). Soluble and insoluble fractions were then separated by centrifugation at 15,000 g for 30 min at 4 °C. The supernatant was loaded into 1 ml HisTrap HP cartridge (GE Healthcare) for purification of His-tag eluted BuIFN-T protein through IMAC. After equilibration of column with his binding buffer, protein was allowed to bind at a flow rate of 1.0 ml/min and the bound protein was washed with wash buffer consisting of 50 mM sodium phosphate buffer, 0.3 M NaCl, 20 mM imidazole, pH 8.0 (for HisTrap HP column). Adsorbed Histag-BuIFN-T was eluted with an imidazole step wise gradient. A gradient of 0–30% was run in 50 ml of elution buffer consisting of 1 M imidazole, 50 mM sodium phosphate buffer, 0.3 M NaCl, pH 8.0. The purified fractions (i.e. the His elutes containing the recombinant fusion protein), the washing fraction (i.e. fraction collected during washing with 20 mM imidazole wash buffer), unbound fractions along with cell pellet and cell lysate as positive control were subjected to SDS–PAGE. Both the eluted and the washing fraction contained the purified protein as major band but there was difference in the number of contaminating bands. They were then used, separately, for further purification. 2.3.2. Purification by ion exchange chromatography (IEX) For the next purification step, we selected the Q Sepharose Column Chromatography (QSCC), because the isoelectric point of BuIFN-T calculated from the amino acid sequence data was 5.95. The column was equilibrated with 50 mM Tris HCl buffer containing 50 mM NaCl pH 8.0, The HIS elute was loaded on the Q Sepharose column. and the adsorbed proteins were eluted with NaCl step wise gradient. A gradient of 0–50% was run in 50 ml of elution buffer consisting of 50 mM Tris HCl and 1 M NaCl, pH 8.0. The eluted fractions were concentrated and analyzed by SDS PAGE. 2.3.3. Purification by gel exclusion chromatography (GEC) Finally, the QSCC elute and the wash elute of IMAC both were further, concentrated and desalted using Centricon centrifugal filter units of 5 kD membrane (Millipore, USA) and were loaded to 1.6  60 cm Sephacryl S-100 (GE healthcare) column in 50 mM phosphate buffer containing 0.15 M NaCl pH 7.0 at a flow rate of 0.5 ml/min. The eluted fractions were concentrated and subjected for SDS PAGE analysis. 2.4. Western blot The purified recombinant BuIFN-T protein, eluted by GEC, was confirmed by western blot analysis and quantified using Bradford assay. For western blot, the membrane was incubated with anti 6-His Epitope Tag mice Antibody (Pierce, Thermo scientific) at 1:1000 dilutions. The secondary antibody was horseradish peroxidase-conjugated Goat Anti-Mouse IgG (Hand L) (Abnova, Thermoscientific) used at 1:2000 dilution. Immunosignal was detected by using DAB system (Bangalore Genei, India). 2.5. In-gel digestion and mass spectrometry (MS) After the confirmation of purified rBuIFN-T protein by western blot, it was subjected to In-gel digestion for mass spectrometric characterization using ESI-LC-MS/MS. The purified rBuIFN-T protein was digested into peptides following in-gel digestion protocol as described by [34]. Electrospray mass spectrometry was performed on maxis HD Impact mass spectrometer (Bruker Daltonics, Germany) with sample introduction from an nano advance UHPLC system (Bruker Daltonics, Germany). Protein digests were eluted from a C18 column with an increasing gradient of acetonitrile with mobile phase containing 0.1% formic acid at 800 nl/min.

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2.6. Antiviral assay of recombinant BuIFN-T Antiviral assay was carried out on Vero cell line challenged with Japanese encephalitis (JE) virus following the protocol standardized in our lab. The cells were cultured in 96-well plates in DMEM medium supplemented with 10% FBS. The culture was monitored till the cells reached 70–80% confluence. The whole assay was done in three groups at five fold serial dilution of pure rBuIFN-T and 0.1, 0.01 and 0.001 multiplicity of infection (m.o.i.) of JE virus in duplicates with appropriate positive and negative controls (Fig. 1). First group was exposed to five fold serial dilutions of rBuIFN-T for 12 h followed by addition of 0.1– 0.001 m.o.i. JE virus (a). The second group was first exposed to 0.1–0.001 m.o.i. JE virus together with five fold serial dilutions of rBuIFN-T (b). The last group was first exposed to 0.1–0.001 m.o.i. JE virus for 12 h, followed by addition of fivefold serial dilutions of rBuIFN-T (c). All of them were incubated overnight at 37 °C. Control samples included only cells without virus/with only virus, without rBuIFN-T/with only rBuIFN-T with equivalent volume being maintained with maintenance media. After 24 h of the beginning of the experiment, cells were observed intermittently under an inverted microscope for plaque formation to measure the cytopathic effect (CPE) [35]. The highest dilution of recombinant BuIFN-T showing 50% reduction in CPE induced by JE virus was noted and used for the determination of specific activity. For calibration of the assay against bovine interferon tau (My biosource, MBS836704) standard reference, same procedure was followed taking bovine interferon tau as the reference protein in place of BuIFN-T. 2.7. Real time analysis/relative expression of BuIFN-T transcripts across different stages of in vitro buffalo embryos For real time PCR, RNA was extracted from six invitro produced embryos (of each stage) using RNAqueousÒ-Micro kit (Cat. No. #AM1931) following the manufacturer’s instruction. The RNA so extracted was reverse transcribed with M-MuLV reverse transcriptase (#K1621, Fermentas Corp, USA), oligo (dT) primer, and 10 mM each of dNTP mix at 42 °C for 60 min. Real-time PCR of the transcripts of BuIFN-T was carried out on different stages of in vitro buffalo embryos using LC-480 light cycler (Roche, Germany). The primer sequence for BuIFN-T was as: forward primer-50 GGAGAC TCATGCTGGATGCC30 ; reverse primer- 50 CCATCTCCTGGGGAA GACCA30 giving a product length of 114 bp (GenBank: JX481983.1). GAPDH was used as the house keeping gene [36]. The level of BuIFN-T transcript on 8–16 cell embryo was used as the calibrator. The reaction mixture comprised 10 ll reaction volume containing 5 ll of SYBER Green qPCR master-mix (#K0251, Thermo Scientific, USA), 0.2 ll of 10 lM of each primer, and 2diluted c-DNA. Thermal cycling conditions consisted of initial denaturation at 95 °C for 5 min, followed by 40 cycles of 15 s at 95 °C, 30 s at 63 °C and 30 s at 72 °C followed by 95 °C for 10 s. All primer pairs used were confirmed for their PCR efficiency, and specific products were checked by melt curve analysis and for the appropriateness of size by 2% agarose gel electrophoresis. The expression data were normalized to the expression of housekeeping genes. The relative expression of the BuIFN-T was determined by 2DDCT [37]. The experiment was carried out in three biological and two technical replicates respectively. 2.8. Effect of recombinant BuIFN-T on in vitro development of buffalo embryos 2.8.1. In vitro maturation of oocytes Oocytes were collected from buffalo ovaries obtained from slaughter house in antibiotic-fortified warm saline, by aspiration

Please cite this article in press as: Saugandhika S et al. Expression and purification of buffalo interferon-tau and efficacy of recombinant buffalo interferontau for in vitro embryo development. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.03.012

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Fig. 1. Antiviral assay of recombinant Buffalo interferon-tau: The whole assay was done in three groups viz. rBuIFN-T exposure followed by virus infection after 12 h (a), simultaneous virus infection and rBuIFN-T exposure (b) and virus infection followed by rBuIFN-T exposure after 12 h (c). Each group was subjected to 0.1, 0.01 and 0.001 m.o.i. of JE virus in duplicates, with five fold serial dilution of pure rBuIFN-T. Appropriate positive and negative controls for CPE were also used.

of surface follicles with 18 gauge needle attached to a 10 ml syringe containing the aspiration medium (TCM-199 + 0.3% BSA + 50 lg/ml gentamicin sulfate). Oocytes with unexpanded multilayered cumulus mass and homogeneous cytoplasmic granulation were subjected to in vitro maturation (IVM) in 100 ll droplets (10–15 oocytes/droplet) of IVM medium (TCM-199 + 10% FBS + 5 lg/ml pFSH + 1 lg/ml estradiol + 0.81 mM sodium pyruvate + 50 lg/ml gentamycin sulphate) for 24 h in a CO2 incubator (5% CO2 in air) at 38.5 °C.

due to addition of rBuIFN-T, equal amount of DPBS was added to the media in the control group corresponding to the amount of recombinant BuIFN-T added in the treatment group as the recombinant protein was reconstituted in DPBS medium. The IVC medium was changed with fresh medium containing respective concentrations of rBuIFN-T at 48 and 96 h post insemination. The cleavage rate was recorded at 48 h post insemination whereas the embryonic development was examined at days 3, 5, 7, 8 and 9 post insemination.

2.8.2. Sperm preparation and in vitro fertilization Sperm capacitation was carried out as described earlier [38]. Briefly, two straws of frozen thawed ejaculated buffalo semen were washed twice with the washing Bracket and Oliphant (BO) medium (BO medium containing 10 lg/ml heparin, 137.0 lg/ml sodium pyruvate and 1.942 mg/ml caffeine sodium benzoate). The pellet was resuspended in around 0.5 ml of the washing BO medium. The in vitro matured oocytes were washed twice with the washing BO medium and transferred to 50 ll droplets (15–20 oocytes/droplet) of the capacitation and fertilization BO medium (washing BO medium containing 10 mg/ml fatty acid-free BSA). 50 ll of the spermatozoa, in capacitation and fertilization BO medium (2–4 million spermatozoa/ml), were then added to the droplets containing the oocytes, covered with sterile mineral oil and placed in a CO2 incubator (5% CO2 in air) at 38.5 °C for 18 h for IVF.

2.8.4. Experimental design for supplementation of IVC medium with rBuIFN-T A total of 240 presumed zygotes, of each trial, were randomly divided into 4 groups and were cultured in IVC medium supplemented with 0 (control), 1, 2 or 4 lg/ml recombinant BuIFN-T. The experiment was repeated 3 times.

2.8.3. In vitro culture After 18 h of sperm-oocyte incubation, the cumulus cells were washed off the presumed zygotes by gentle pipetting. The presumed zygotes were then divided into four different groups (control, 1, 2 and 4 lg/ml recombinant BuIFN-T, the concentrations of IFN-T were chosen as per [29] except for 0.2 lg/ml which was replaced by 1 lg/ml) and washed several times with modified Charles Rosenkrans medium with amino acids (mCR2aa) containing 0.8% BSA and different concentrations of recombinant BuIFN-T and were cultured in this medium for up to 9 days post insemination. To prevent dilution of media in the treatment group

2.8.5. Statistical analysis The cleavage rate and blastocyst formation rate (% data) were analyzed using SYSTAT 7.0 (SPSS Inc., USA) after arcsin transformation of percentage values. To find out if recombinant BuIFN-T has any effect on in vitro development of embryos, the percentage of embryos that developed to various embryonic stages at days 5, 7, 8 and 9 post insemination were compared between treatment groups and their respective controls. The differences were analyzed by one way analysis of variance (ANOVA) followed by Fisher’s LSD test. 2.8.6. Blastocyst cell count Total cell numbers of day 9 blastocysts were counted after Hoechst 33342 staining. The nuclei of 20–25 IVF blastocysts were stained with Hoechst 33342 (10 ng/ml) for 10 min and then washed 3 times in Dulbecco’s PBS. The embryos stained with Hoechst 33342 were individually mounted on microscope slides in glycerol beneath a cover slip and examined as a whole-mount preparation under UV.

Please cite this article in press as: Saugandhika S et al. Expression and purification of buffalo interferon-tau and efficacy of recombinant buffalo interferontau for in vitro embryo development. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.03.012

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3. Results 3.1. Cloning of in BuIFN-T in pJET cloning vector High quality total RNA was successfully isolated from in vitro produced buffalo blastocysts (Fig. 2A) and reverse transcriptase PCR (RT-PCR) reaction of the total RNA was carried out under optimum conditions. The products obtained from the RT-PCR reaction were resolved on a 1.5% agarose gel electrophoresis with ethidium bromide staining. A distinct band of approximately 694 bp in length was successfully amplified (Fig. 2B). The positive clones with the DNA insert of interest on sequencing revealed the inserted DNA to be 694 bp in length corresponding to the estimated size determined by agarose gel electrophoresis and matched with the published sequence of BuIFN-T1 from GenBank (accession No. JX481984) against which the primers were designed. 3.2. Expression construct pET22b-BuIFN-T The BuIFN-T gene cloned in expression vector pET22b, was confirmed by PCR giving an expected band of 525 bp on agarose gel and further validated by restriction digestion of the recombinant vector pET22b-BuIFN-T which released amplicon of 525 bp size (Fig. 2C). Sequencing of recombinant plasmid revealed intact contiguous ORF of BuIFN-T through His-tag suggesting that the expression vector was ready to express BuIFN-T (Supplementary File b). 3.3. Expression of recombinant BuIFN-T (rBuIFN-T) To produce recombinant IFN-T, we initially transformed the recombinant plasmid pET22b-BuIFN-T into Top 10 cells and on

Fig. 2. Agarose gel electrophoresis of total RNA isolated from in vitro produced buffalo blastocyst (A), PCR amplified product of BuIFN-T with signal sequence (694 bp) (B), recombinant BuIFN-T (without signal sequence) released by restriction digestion of the recombinant vector pET22b BuIFN-T from TOP10 cells positive colonies (525 bp) (C), screening of recombinant colonies of BL21(DE3) cells transformed with recombinant vector pET22b BuIFN-T (D).

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analysis of the insert sequence ORF from positive clones (Fig. 2D), recombinant plasmid was isolated and transformed into E. coli BL21(DE3) strain (Fig. 2D). Transformed BL21 E. coli cells when induced at 37 °C and 25 °C with 1 mM, IPTG, resulted in production of 20 kDa protein in the cell pellet fraction (Fig. 3). The experiment revealed very low yield of recombinant BuIFN-T in soluble fraction. Thus, to improve the solubility of the recombinant BuIFN-T, when 1.5 ml of transformed BL21(DE3) E. coli cells were induced at lower temperatures of 16 °C and 12 °C, the proportion of recombinant BuIFN-T in soluble fraction increased to approximately 60–70% (Fig. 4) as confirmed by SDS PAGE. 3.4. Purification of recombinant BuIFN-T The rBuIFN-T produced in E. coli cultures was successfully purified to homogeneity by sequential chromatographic method. Immobilised Metal Affinity Chromatography (IMAC) based affinity purification using His-tag at the C terminal of rBuIFN-T resulted in partial purification of rBuIFN-T (Figs. 5 and 6). Purification of the rBuIFN-T through IMAC resulted two broad fractions- the eluted fraction during gradient of 300 mM Imidazole and the washing fraction (i.e. fraction collected during washing with 20 mM Imidazole wash buffer). As shown in Fig. 5 both fractions contained high amount of 20 kDa expressed protein. Interestingly, washing fraction had very low level of contaminant proteins in comparison to eluted fraction. Eluted fraction was further purified by ion-exchange chromatography (IEX) using Q Sepharose Column. After Q Sepharose Column Chromatography QSCC, the eluted fraction was highly pure showing only single contaminating band at 40 kDa, in addition to our expressed recombinant protein (20 kDa), as evident from SDS PAGE (Figs. 5 and 6). To remove the contaminating 40 kDa protein, the Q Sepharose column elute was purified by Gel Exclusion Chromatography (GEC) using Sephadex G-100. The washing fraction obtained from IMAC affinity purification was also purified by GEC using Sephadex G-100. GEC resulted in two peaks of the elution protein and SDS–PAGE analysis of the latter peak, finally, resulted in single band of 20 kDa rBuIFNT protein (Figs. 5 and 6). The western blot analysis using anti his mouse antibody and horseradish peroxidase HRP conjugated secondary antibody with Di-amino benzene (DAB) system as substrate showed a fine single interaction at 20 kDa location confirming that

Fig. 3. SDS PAGE analysis showing expression of rBuIFN-T (20kD) in BL21(DE3) cells induced at 37 °C and 25 °C with 1 mM, IPTG: Lane 1 – uninduced control, Lane 2 – rBuIFN-T in cell pellet fraction at 25 °C, Lane 3 – soluble fraction at 25 °C, Lane 4 – Marker, Lane 5 – rBuIFN-T in soluble fraction at 37 °C, Lane 6 – rBuIFN-T in cell pellet fraction at 37 °C. rBuIFN-T protein was present at high concentration in cell pellet fraction and low concentration in soluble fraction at both temperature, 25 °C and 37 °C.

Please cite this article in press as: Saugandhika S et al. Expression and purification of buffalo interferon-tau and efficacy of recombinant buffalo interferontau for in vitro embryo development. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.03.012

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Fig. 4. SDS PAGE analysis showing expression of rBuIFN-T (20kD) in BL21(DE3) cells induced at 12 °C and 16 °C with 0.5 mM, IPTG: Lane 1 – marker, Lane 2 – uninduced control, Lane 3 – rBuIFN-T in cell pellet fraction at 12 °C, Lane 4 – soluble fraction at 12 °C, Lane 5 – rBuIFN-T in cell pellet fraction at 16 °C, Lane 6 – soluble fraction at 16 °C. rBuIFN-T protein was present at high concentration in soluble fraction and low concentration in cell pellet fraction at both temperature, 12 °C and 16 °C.

the purified protein was rBuIFN-T and there was no contaminating protein in the preparation (Fig. 7). 3.5. Liquid chromatography mass spectroscopy (LC-MS) confirmation of the purified protein rBuIFN-T Identification of the pure protein by LC-MS search resulted in top hits of IFN-T with very high score (4850). This indicates that purified rBuIFN-T protein is highly pure (Supplementary File a).

Similarly, in group 3, the subgroup which was exposed to 0.01 m.o.i. of JE virus followed by 5-fold serial dilution of rBuIFNT protein after 12 h, 5th dilution of rBuIFN-T (0.027  104 mg of rBuIFN-T) showed 40% reduction in CPE after 24 h (Fig. 8C). 0.01 multiplicity of infection (m.o.i.) of JE virus induced CPE after 24 h (positive control, Fig. 8D) while 0.1 m.o.i. induced CPE at 12 h and 0.001 m.o.i. did not induce CPE till 24 h. Control sample supplemented with 5th dilution of rBuIFN-T protein (0.027  104 mg of rBuIFN-T) did not induce CPE till 24 h (negative control, Fig. 8E). The 5th dilution of rBuIFN-T (0.027  104 mg of rBuIFN-T) was the highest dilution showing 50%, 70% and 40% reduction in CPE in all three groups – 1, 2 and 3 when inoculated with 0.01 m.o.i. of JE virus. Therefore antiviral activity of rBuIFN-T at an inoculation dose of 0.01 m.o.i. of JE virus was considered appropriate for the antiviral assay. The total amount of protein at the highest dilution exhibiting 50% reduction in JE virus induced CPE was 0.027  104 mg of rBuIFN-T, which yielded 0.037  107 antiviral units per mg of protein in parallel to the standard reference protein. 3.7. Relative expression of BuIFN-T transcripts across different stages of in vitro buffalo embryos BuIFN-T transcripts were detected at 8–16 cell stage and continued in morulae, blastocyst and hatched blastocyst with the highest level of expression in hatched blastocyst stage embryos produced through IVF. There was no significant difference in the relative expression of BuIFN-T transcripts between 8–16 cell stage and morulae stage buffalo embryos. However, the relative expression of BuIFN-T transcripts increased significantly in the blastocyst stage (1.8 folds), which further increased significantly (being highest 2.9 folds) in the hatched blastocyst stage (Fig. 9).

3.6. rBuIFN-T exhibits antiviral activity

3.8. Effect of recombinant BuIFN-T (rBuIFN-T) produced by an E. coli Expression system on in vitro development of buffalo embryos

0.01 multiplicity of infection (m.o.i.) of JE virus induced CPE after 24 h which was selected as reference m.o.i. for the CPE inhibition assay study. The rBuIFN-T protein exhibited antiviral activity in all the three groups. In group 1, the subgroup which was exposed to 5-fold serial dilution of rBuIFN-T protein followed by 0.01 m.o.i. of JE virus after 12 h, 5th dilution of rBuIFN-T (0.027  104 mg of rBuIFN-T) showed 50% reduction in CPE after 24 h (Fig. 8A). In group 2, the subgroup that was exposed to 0.01 m.o.i. of JE virus and 5-fold serial dilution of rBuIFN-T protein together, 5th dilution of rBuIFN-T (0.027  104 mg of rBuIFN-T) showed 70% reduction in CPE at the end of 24 h (Fig. 8B).

To find out the impact of rBuIFN-T on embryo cleavage and blastocyst formation, the presumptive buffalo zygotes, after in vitro fertilisation (IVF), were randomly assigned to the control or rBuIFNT treatment groups. Table 1 shows the effect of different concentration of rBuIFN-T on the development of buffalo embryos. Out of the three concentrations of rBuIFN-T (1 lg/ml, 2 lg/ml and 4 lg/ml), the blastocysts development rate (blastocysts development rate is calculated on the cleaved embryos) at concentration of 2 lg/ml was significantly higher than control (45.55 ± 5.1 vs. 31.11 ± 1.1, P < 0.01) and even the number of cells within the resulting blastocysts, cultured in 2 lg/ml rBuIFN-T, was found to

Fig. 5. SDS PAGE analysis of; (A) IMAC based affinity Chromatography: Lane 1 – marker (M), Lanes 2 and 3 (positive control) – rBuIFN-T in cell pellet fraction (P) and soluble fraction (L), Lane 4 – partially purified rBuIFN-T in HIS eluted fraction (HE) and Lane 5 – partially purified rBuIFN-T in washing fraction (WF); (B) ion Exchange Chromatography (IEX): Lane 1 – Marker (M), Lane 2 (positive control) – rBuIFN-T in cell pellet fraction (P), Lanes 3, 4, 5, 6 and 7 – partially purified rBuIFN-T in QSCC elutes; and (C) Gel Exclusion Chromatography (GEC): Lane 1 – marker (M), Lane 2 – positive control (P) – rBuIFN-T in cell pellet fraction, Lanes 3, 4, 5, 6, 7 and 8 – completely purified rBuIFN-T in GEC elutes.

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Fig. 6. Chromatogram of; (A) IMAC based affinity purification: purification of the rBuIFN-T through IMAC resulted two broad fractions – His elute (HE) i.e. the fraction that eluted during gradient of 300 mM Imidazole and the washing fraction (WF) i.e. fraction collected during washing with 20 mM imidazole wash buffer. Both the fractions contained partially purified rBuIFN-T; (B) Ion-Exchange Chromatography: Purification of the rBuIFN-T through IEX resulted elution of partially pure QSCC elute during a gradient of 50% (run for 50 ml), in a shouldered peak manner which had a single contaminating band at 40 kDa along with pure rBuIFN-T; and (C) Gel-Exclusion Chromatography: purification of the rBuIFN-T through GEC, resulted in two peaks of the elution protein with elution of highly pure rBuIFN-T in the latter peak.

than that of control (21.41 ± 1.62 vs. 14.2 ± 0.48, P < 0.05) (Table 1, Fig. 10). However, rBuIFN-T at 2 lg/ml concentration, failed to affect cleavage rate significantly compared to control (47.5 ± 2.25 vs. 42.2 ± 1.13). At the concentration of 1 lg/ml, rBuIFN-T had no significant effect either on the cleavage rate (42.2 ± 1.13 vs. 39.07 ± 1.8) or on the rate of blastocyst development (31.11 ± 1.11 vs. 26.97 ± 1.8) as compared to control. Exposure of the presumed zygotes to recombinant BuIFN-T at concentration of 4 lg/ml, reduced (P < 0.01) the cleavage rate compared to control (42.2 ± 1.13 vs. 31.11 ± 1.11, Table 1). At this concentration, out of the 48 cleaved embryos subjected to in vitro culture (IVC), only 5 developed to blastocyst stage. 3.9. Hoechst staining result Fig. 7. Western blot analysis of rBuIFN-T using anti-his mouse antibody and HRP conjugated secondary antibody with DAB system as substrate showed a fine single interaction at 20 kDa location confirming the purity of rBuIFN-T.

be greater than the control (189 ± 12.68 vs. 109.75 ± 7.5, P < 0.05) (Table 1 and Fig. 10). Moreover the number of hatched blastocysts in 2 lg/ml treatment group on Day 9 was also significantly greater

Total cells of day 9 blastocysts derived from control, IVC medium supplemented with 2 lg/ml rBuIFN-T and IVC medium supplemented with 1 lg/ml of BuIFN-T were counted after Hoechst 33342 staining. It was found that there were more number of cells in blastocysts cultured in IVC medium supplemented with 2 lg/ml of rBuIFN-T compared to blastocysts in control (189 ± 12.68 against 109.75 ± 7.5) while there were lesser number of cells in blastocysts cultured in IVC medium supplemented with 1 lg/ml

Fig. 8. Antiviral assay of recombinant Buffalo interferon-tau: 0.01 m.o.i of JE virus along with 5-fold serial dilution of rBuIFN-T was subjected to three main test groups viz. rBuIFN-T exposure followed by virus infection after 12 h (A), simultaneous virus infection and rBuIFN-T exposure (B) and virus infection followed by rBuIFN-T exposure after 12 h (C). Positive control (JE virus without rBuIFN-T) (D), negative control (rBuIFN-T without JE virus) (E). The reduction in cytopathic effects shown by test groups A, B and C at 0.01 m.o.i and 0.027  104 mg of rBuIFN-T was 50%, 70% and 40% respectively.

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Fig. 9. Relative expression of BuIFN-T transcripts across different stages of in vitro developed buffalo embryos: expression of BuIFN-T transcripts was found at 8–16 cell stage, which continued in morulae, blastocyst and hatched blastocyst with the highest level of expression in hatched blastocyst. ⁄, ⁄⁄ and ⁄⁄⁄ shows significance of variation among the groups (P < 0.05). Groups marked with different stars have significant differences in the relative expression of BuIFN-T.

concentration of rBuIFN-T compared to blastocysts in control (84.25 ± 3.75 against109.75 ± 7.5) (Table 1 and Fig. 10).

4. Discussion The present study is about the strategies to produce rBuIFN-T in bacterial cells followed by its purification to homogeneity, identification, test of biological activity and finally, its impact on in vitro development of blastocyst rate in buffalo. IFN-T has been recognized as the key molecule in maternal recognition of pregnancy (MRP) and uterine receptivity for conceptus growth in ruminants [3,5,39]. Recently a few studies suggest the growth promoting effects of IFN-T on the in vitro development of preimplantation embryos [31,40]. In the present study we found that IFN-T mRNA transcripts were present as early as 8–16 cell stage which continued to express till hatched blastocyst stage in in vitro produced buffalo embryos. Inquisitively, we attempted to test the role of BuIFN-T on cleavage and blastocyst formation. Native purification of IFN-T is challenging because of its transient secretion in a very short time window (16–25 days) of pregnancy in buffaloes. IFN-T is a small molecular weight protein of around 18–20 kDa which exists in multiple isoforms [41]. In our earlier work BuIFN-T1 isoform had been identified as the relatively predominant isoform from rest isoforms at transcript level (unpublished data) and here in the present study BuIFN-T1 isoform was used for recombinant expression and purification studies. We produced recombinant clones of BL21(DE3) E. coli cells harboring pET22b-BuIFN-T and optimized the conditions for large scale expression of recombinant BuIFN-T in soluble state. It was essential because bacterial expression often ends up with larger share of protein in insoluble aggregate [26]. In this study we achieved higher solubility of rBuIFN-T by step wise decrease of the induction

temperature. Transformed BL21(DE3) cells when induced at 25 °C there was hardly any significant increase in solubility (Fig. 2E). As the temperature was decreased to 16 °C, expression in soluble fraction increased and the maximum expression in soluble state was obtained when incubated at 12 °C for 22 h with 0.5 mM IPTG (Fig. 2F). The expressed protein has a tag of 6 His residues at the C-terminal which was used for first step purification using IMAC column which yielded around 1805 lg of total protein from 1 l of bacterial culture. Affinity purification revealed contamination of rBuIFN-T with some higher size proteins at 40 kDa band and 30 kDa which was removed by IEX (ion exchange chromatography) and GEC. Analysis of GEC purified fractions on 15% SDS PAGE showed two bands, with the second peak/band containing functionally active rBuIFN-T. Eventually, it was found that from 1 l of culture 1216 lg of highly pure rBuIFN-T could be obtained. SDS PAGE and western blot results showed that the purified protein is around 20 kDa in size and highly pure, as evident from the single band seen in SDS PAGE (Figs. 5–7). Homogeneity of the purified protein was further confirmed by LC-MS mass spectrometer, which clearly indicated that the purified rBuIFN-T is 99% pure (Supplementary File b). Further, to characterize the biological activity of pure rBuIFN-T, antiviral assay on JE virus was done which revealed that the protein was able to inhibit CPE up to 50% at a concentration of 0.01 lg/ll. In our study the specific antiviral activity of rBuIFN-T was found to be 0.037  107 units/ mg. Previous workers have estimated the biological activity of such preparations of IFN-T on different antiviral assay system and found different results [24–26]. Cleavage of embryo leading to blastocyst followed by its hatching is a complex event which depends on multiple factors. In this regard, in vitro media supplementation of hormones or cytokines, governing a specific event, becomes a critical factor for increasing the viability/blastocyst production rate of in vitro embryo development [42,43]. In the present study, we found that IFN-T transcripts are present as early as 8–16 cell stage of in vitro embryo development which insinuates toward a novel role of IFN-T which may be different from MRP. In MRP, IFN-T acts on uterine cell receptors in paracrine fashion, while expression of BuIFN-T ahead of MRP may be an indication of its action in autocrine fashion. A recent research revealed that IFN-T acts as an autocrine factor to regulate ovine trophectoderm cell proliferation [32]. Recent studies report that recombinant bovine IFN-T derived from both baculovirus expression system and E. coli expression system at a concentration of 100 ng/ml and 2 lg/ml respectively promoted early in vitro embryonic development [31,40]. Moreover, earlier studies indicate that bovine embryos develop rapidly when they are cocultured with trophoblastic vesicles produced from elongating conceptuses of day 14 pregnancy [44–46]. The overall findings aroused our curiosity to know if IFN-T has any impact on in vitro blastocysts development. We decided to test three doses of rBuIFN-T, a lower dose of 1 lg/ml, a medium dose of 2 lg/ml and a higher dose of 4 lg/ml on blastocyst development rate and found that rBuIFN-T at a concentration of 2 lg/ml, supplemented in the IVC medium, not only augmented the blastocyst formation rate in vitro but also increased the

Table 1 Effect of recombinant Buffalo Interferon-Tau (BuIFNT) on the development of in vitro fertilized buffalo embryos. Conc. Of rBuIFNT:(lg/ml)

No. of Oocytes (n)

Cleavage at day 3: n (%)

Morula at day 5: n (%)

Blastocyst at day 8: n (%)

Hatched blastocyst at day 9: n (%)

No of blastocysts for cell counting

Blastocyst cell number ± SEM

0 (Control) 1 2 4

168 158 155 151

71 (42.2 ± 1.13) 62 (39.07 ± 1.83) 74 (47.51 ± 2.25) 48 (31.6 ± 2.91)

40 (54 ± 7.8) 26 (41.61 ± 4.2) 44 (60.2 ± 4.32) 20 (41.66 ± 1.6)

22 17 33 5

10 (14.2 ± 0.48) 6 (9.36 ± 1.74) 16 (21.41 ± 1.62)a

4 4 4

109.75 ± 7.5 84.25 ± 3.75 189 ± 12.68a

(31.11 ± 1.11) (26.97 ± 1.8) (45.55 ± 5.16)a (10.18 ± 0.92)

Data from three trials. n Means total number and data in parenthesis shows mean % ± SEM. a Superscripts indicate the significant increase in the treatment group as compared to control (P < 0.05).

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Fig. 10. Hatched blastocysts in IVC medium supplemented with 2 lg/ml of rBuIFN-T (A). Hatched blastocyst, expanded blastocyst, early blastocyst in control (B). Hoechst staining of blastocyst derived from control (C), IVC medium supplemented with 2 lg/ml of rBuIFN-T (D) and IVC medium supplemented with 1 lg/ml of rBuIFN-T (E). There was an increase in number of cells within blastocysts cultured in IVC medium supplemented with 2 lg/ml concentration of rBuIFN-T as compared to control and IVC medium supplemented with 1 lg/ml concentration of rBuIFN-T. Quantification of Hoechst staining for figure-C–E has been done by Image J software (imagej.nih.gov/ij) (Supplementary File c).

total number of cells within the resulting blastocysts. Moreover there was also a significant increase in the number of hatched blastocyst at day 9 post insemination. But neither the lower concentration of 1 lg/ml nor the higher concentration of 4 lg/ml, showed any increase in the rate of blastocyst formation. On contrary, the higher dose diminished the rate of blastocyst development. Coming out with an optimized doze of IFN-T in in vitro experiment is very complex. There are several reports suggesting antiproliferative activity of type1 IFNs like IFN-a and IFN-T on cell growth [16,47]. A few studies revealed that the antiproliferative activity of IFN-T is cell specific [48]. Probably, it may be because of antiproliferative role of IFN-T at higher concentration that 4 lg/ml concentration of rBuIFN-T inhibited embryonic development to blastocyst stage. Any information on the level of IFN-T in early stages of embryonic development will be very important as it may play a decisive role in the medium supplementation of embryo culture with IFN-T, because supplementation of BuIFN-T at a higher level than required may prove detrimental to embryonic development. An earlier study reported that IFN-T had no negative effects on the development of mice embryos in vitro [49]. Survival and implantation rates of the mouse embryo are promoted by perfusion of IFN-T into the abdominal cavity [50]. IFN-T exerts its biological activities by binding to the type I interferon receptor (IFNAR), which consists of 2 transmembrane chains, IFNAR1 and IFNAR2 [51,52,30]. Previously, IFNT receptor was thought to be expressed only in the endometrium [52] and not in conceptuses until at least 15 d of pregnancy. However, later few reports demonstrate the expression of IFNAR1 at earlier stages in ovine conceptuses [53] and in bovine embryos from the morula to blastocyst stage [31]. Thus it may be possible that the beneficial effect of IFN-T on in vitro embryo development is through the effects of IFN-T receptor acting in an autocrine manner [40]. Two different studies report that GJA1 and CDH1 are two genes playing crucial role in blastocyst formation [54,55]. CDH1 gene encoding for Ecadherin is one among the specific set of genes that direct the acquisition of cell polarity within the trophectoderm which drives for blastocyst formation [55]. In addition GJA1 gene that encodes

connexin 43 is crucial for the maintenance of compaction in bovine embryos [54]. A recent study examined the effect of recombinant bovine IFN-T on in vitro developed bovine embryos and relative concentration of GJA1 and CDH1 genes in in vitro developed bovine embryos cultured in recombinant bovine IFN-T. It reported that the recombinant bovine IFN-T enhanced in vitro development of bovine embryos by upregulating the expression of connexin 43 and E-cadherin proteins which are expressed both at mRNA and protein level in the in vitro developed embryos [40]. However, the detailed mechanism by which rBuIFN-T is augmenting the blastocyst development rate at a concentration of 2 lg/ml is further researchable. 5. Conclusion Earlier studies [56,25] reported the expression of bovine IFN-T in E. coli, where the protein was produced as insoluble aggregates. To the best of our knowledge for the first time recombinant BuIFNT was purified to homogeneity from prokaryotic expression system having biological activity as evident from antiviral assay. Further, purified recombinant BuIFN-T at concentration of 2 lg/ml, also improved in vitro blastocyst development rate when supplemented in the IVC medium. However, for higher yield of rBuIFN-T, large scale purification may be optimized in eukaryotic expression systems like P. pastoris. As in vivo and in vitro reproductive performance of buffalo is lagging behind other species, rBuIFN-T may improve in vitro embryo production rate and in vivo embryo transfer efficiency and may also have a research application in human disease models. Acknowledgements This research was funded by Indian Council of Agricultural Research (ICAR), Government of India. We would also like to thank the Council of Scientific and Industrial Research (CSIR), India for providing the financial assistance to the student in the form of senior research fellowship. The authors declare that there is no

Please cite this article in press as: Saugandhika S et al. Expression and purification of buffalo interferon-tau and efficacy of recombinant buffalo interferontau for in vitro embryo development. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.03.012

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conflict of interest that would prejudice the impartiality of this scientific work.

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Appendix A. Supplementary material

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cyto.2015.03.012.

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Please cite this article in press as: Saugandhika S et al. Expression and purification of buffalo interferon-tau and efficacy of recombinant buffalo interferontau for in vitro embryo development. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.03.012