Cloning and expression of Iranian Turkmen-thoroughbred horse follicle stimulating hormone in Pichia pastoris

1. Background
    Superovulation in horses is much less successful than in other domestic species. In addition to superovulation rate, the number of embryos are typically negligible (1). Hence, there is a great demand to develop products that induce multiple ovulations in mares due to changes in the governance of the horse reproduction industry (2). Follicle stimulating hormone (FSH), a member of the glycoprotein hormone family, plays an essential role in this process (3). Generally, the anterior pituitary gland is the tissue that is responsible for releasing this hormone (4-8), and granulosa cells in the ovary are the target cells for the numerous actions of FSH (6, 8-11). Similar to other members of the glycoprotein hormone family, including luteinizing hormone (LH), chorionic gonadotropin hormone (CGH), and thyroid stimulating hormone (TSH), FSH consists of two heterodimer non-covalently linked subunits: a common alpha subunit and a hormone-specific beta subunit (6, 12). An increase in the exogenous FSH concentration in the circulatory system during follicular development may leads to increase in the number of ovulatory follicles, which results in an increase in the number of mature follicles, the ovulatory rate, and the rate of embryo transfer (3). Currently, this method is applicable for both human infertility and animal breeding programs(13). Some gonadotropin sources with FSH-like activity, such as pituitary extraction from domestic animals, pregnant mare serum gonadotropin (PMSG), and FSH extraction from the urine of postmenopausal women, have been used for the same purpose. Despite the success of some of these treatments in many domestic species, these sources are less effective in mares (14-18). It has been reported that recombinant eFSH (reFSH) shows a much more specific effect in increasing the number of total preovulatory follicles than other therapeutic methods (3). Many of the recombinant proteins that have been successfully produced using the yeast expression system have been used in basic laboratory research and the therapeutic industry (19). The methylotrophic P. pastoris yeast is a suitable host for the production of many recombinant proteins because of its easy genetic manipulation, expression of intracellular or extracellular foreign protein (20), and capability to perform eukaryotic protein modifications, such as glycosylation, disulfide bond formation, and proteolytic processing (20-22).

2. Objectives
    The purpose of the present research was to express the recombinant horse FSH using the P. pastoris expression system and to detect this protein in the secretion media. This study describes the first production of the horse FSH in the yeast expression system using the Iranian native mare fsh gene.

3. Materials and Methods
    The chemical and biological laboratory reagents and instruments that were used in this study include NucleoSpin RNA kit (Macherey Nagel, Düren, Germany); Revert AidTM H minus first-strand cDNA synthesis kit (Fermentas, Massachusetts, USA); pTZ57R/T vector (Fermentas, Massachusetts, USA); DNA sequencing analysis (MWG, Ebersberg, Germany); Pichia expression kit, (Invitrogen, Massachusetts, USA); P. pastoris cell culture media (Invitrogen, Massachusetts, USA); Gene-Pulser (Bio-Rad, callifornia, USA); sorbitol (Invitrogen, Massachusetts, USA); Amplicon Ultra 3000 MWCO (Millipore Bedford, Massachusetts, USA); Primary antibodies against the eFSHa and eFSHb subunits  (Santacruz, Texas, USA); horse radish peroxidase (HRP)-conjugated secondary antibodies against eFSHa and eFSHb subunits (Sigma Aldrich, Missouri, USA); nitrocellulose membranes (Amersham, london, UK); enhanced chemiluminescence (ECL) reagent (Amersham ECL, London, UK); Aautoradiography GBX developer (Sigma Aldrich, Missouri, USA); Amersham Hyperfilm ECL instrument (Amersham, london, UK); and protein G immunoprecipitation kit (Sigma Aldrich, Missouri, USA).

3.1. Strains and Media
    The E. coli DH5α strain and P. pastoris yeast in a Pichia expression kit were used as the competent cells. Luria Bertani (LB) medium (1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 1% (w/v) NaCl, pH 7.0) was used as the bacterial growth culture at 37°C (25). The P. pastoris cell culture media  was composed of yeast extract peptone dextrose (YEPD) medium (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose, and 0.1% (w/v) ampicillin), minimal dextrose (MD) medium (1.34% (w/v) yeast nitrogen base with ammonium sulfate without amino acids (YNB), 4×10-5% (w/v) biotin, 2% (w/v) dextrose, 0.05% (w/v) ampicillin, and 1.5% (w/v) agarose), buffered glycerol-complex (BMGY) medium (1% (w/v) yeast extract, 2% (w/v) Bacto-peptone, 1.34% (w/v) YNB, 1% (v/v) glycerol, 4×10-5% (w/v) biotin, and 100 mM potassium phosphate buffer pH 6.0) and buffered methanol-complex (BMMY) medium, supplemented with 0.5% (v/v) methanol.

3.2. Cloning of the efshα and efshb cDNAs into Expression Vector
    The total mRNA from the Iranian Turkmen-thoroughbred mare’s anterior pituitary gland was freshly extracted using a NucleoSpin RNA kit. The cDNA synthesis was then performed with 1 mg of total mRNA using a Revert AidTM H minus first-strand cDNA synthesis kit and an oligo (dt) primer that, followed by RT-PCR.
    The primers carrying the AvrII site (Table 1) were used to amplify the efsha and efshb cDNA sequences by polymerase chain reaction (PCR).
     The PCR mixture for each subunit consisted of a total volume of 25 mL that included 2 units of Taq DNA polymerase, 2 mL of RT-cDNA, 2.5 mL of 10× PCR buffer, 1 mL of 10 mM dNTP, 1.5 mL of 50 mM MgCl2, and 0.5 mL of 10 pmoles of each forward and reverse primer. The PCR thermocycling steps for efshα and efshb genes were as follow in Table 2.
     The amplified PCR products were separately cloned through the T/A cloning method using the pTZ57R/T vector and frozen DH5a competent cells. The recombinant bacterial colonies were identified by colony PCR, plasmid DNA digestion using AvrII, and DNA sequencing analysis. Ultimately, the gel-purified products were subcloned by ligation into the AvrII site of the linearized pPIC9 plasmid and then transformed into the DH5a competent cells through the standard protocol (23).

3.3. Recombinant P. pastoris Transformants
    As shown in Figure 1, the recombinant pPIC9-eFSHα and pPIC9-eFSHb vectors were linearized with SalI and SacI, respectively (Pichia expression kit). These recombinant vectors were co-transformed (5 mg) into the host P. pastoris GS115 His+ Mut+ cells (containing aox1 gene) by co-electroporation in a Gene-Pulser with a voltage of 1900 V, capacitance of 25 mF, and resistance of 400 ohms (24).
    The yeast transformants were resuspended in 1 M sorbitol, and the cell mixture was then spread and cultured on selective MD medium plates (histidine-restricted media) at 29.5ºC for four days. The single colonies for His+ transformants were inoculated into the YEPD liquid medium and incubated for 48 h at 29.5ºC with 290-rpm agitation. The presence of both efsha and efshb genes in P. pastoris genome was confirmed by PCR using forward and reverse aox1 primers. The pPIC9 vector without any insert and the recombinant pPIC9-eFSHa and pPIC9-eFSHb vectors were used as control groups (Pichia expression kit).

3.4. Secretion of the Recombinant eFSH
    The positive single-recombinant colonies carrying both recombinant pPIC9-eFSHa and pPIC9-eFSHb vectors were selected and cultured in BMGY medium (25 mL) on a rotary incubator for 18 h at 29.5°C with 290-rpm agitation (25) to reach an OD600 value of approximately 3.0 (24). The yeast cells were harvested by centrifugation at 3000 ×g for 5 min and subsequently resuspended and cultivated in BMMY medium (100 mL). The incubation was continued at 29.5ºC for five days. Methanol was added every 24 h to a final concentration of 0.5% to maintain the induction phase. The samples from five consecutive days (days 0 to 5) were collected at 0, 6, 24, 30, 48, 54, 72, 78, 96, 102, and 120 h after induction. The supernatant of the suspension was separated by centrifugation at 13000 ×g for 3 min and stored at -70ºC for use in the biochemical confirmatory tests. The negative controls, which included GS115 (His+ Muts) cells expressing extracellular albumin, GS115 (His+ Mut+) cells expressing intracellular b-galactosidase, and GS115 (His+ Mut+) cells transformed with non-recombinant pPIC9, were cultured and collected in the same manner (Pichia expression kit).

3.5. Western Analysis
    The secretion media from the recombinant P. pastoris GS115 strain (His+ Mut+) and the control groups were concentrated five-fold using an Amplicon Ultra 3000 MWCO at 13000 ×g. The concentrated samples were electrophoresed in 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) apparatuses through the Laemmli method and blotted 16 h overnight onto nitrocellulose membranes. The membranes were incubated in the blocking solution   (1 M TBS-Tween-20 plus 3% non-fat milk) for 2 h with shaking at 22ºC. The primary antibodies against the eFSHa (Sc-292422) and eFSHb (Sc-18222) subunits were diluted at ratios of 1:750 and 1:800, respectively. Each membrane was incubated with a specific primary antibody for 2 h at 22ºC. The membranes were then washed three times with 1 M TBS-Tween-20 solution for 15 min. The corresponding horse radish peroxidase (HRP)-conjugated secondary antibodies against eFSHa (A5420) and eFSHb (A0545) subunits were diluted at ratios of 1:125000 and 1:500000, respectively. Each membrane was incubated with the corresponding specific diluted secondary antibody for 1.5 h at 22ºC. The membranes were washed five times for 15 min and treated with an enhanced chemiluminescence (ECL) reagent to reveal the immunoreactive bands through an equilibration technique using. The signals were detected on an Amersham Hyperfilm ECL instrument.
3.6. Immunoprecipitation
    A protein G immunoprecipitation kit was used to detect the expressed recombinant eFSH in the P. pastoris BMMY medium. This enrichment was accomplished by binding the recombinant eFSH with a primary specific b-subunit antibody followed by the precipitation of the immune complexes with protein G. The concentrated sample obtained 54 h after induction was chosen to confirm the previous experiments. First, 600 mL of the sample was mixed with 0.5 to 1 mg of the primary antibody, and the mixture was shaken for 14 h at 4ºC. After the sample was transferred to a filter column, 30 mL of G-protein was added to the column, and the mixture was shaken for 14 h at 4ºC. Subsequently, the obtained purified recombinant eFSHb sample and the control sample were analyzed using the Western blotting.

4. Results

4.1. Nucleotide Sequence Analysis of efsh Subunits
      After the amplification of the efsha and efshb genes from cDNA (Figure 2), the sequence analysis of these amplified gene fragments showed that the efsha gene had the expected size of 387 base pairs, which confirmed the information included in the GenBank. However, the efshb gene was a fragment with 465 bp. Our sequence analysis was revealed that eFSHb reported here was a bit different (accession no. JX861871) to what was in GenBank at its 3¢-UTR (Figure 3) with 3 more nucleotides within the 413-to-418 (GCC ® GCTGCC) and having a nucleotide substitution at 443 base pair region (TAT instead of TAC).

4.2. Genomic Integration of the pPIC9-eFSHa and pPIC9-e FSHb Vectors
    The recombinant pPIC9-eFSHα and pPIC9-eFSHb vectors were co-inserted randomly into the genome of P. pastoris cells (Pichia expression kit). The agarose gel electrophoresis showed that which of the P. pastoris GS115 (His+ Mut+) transformants containing both recombinant pPIC9-eFSHα and pPIC9-eFSHb vectors (Figure 4). Based on the standard protocol (Pichia expression kit), the expected size of the P. pastoris genomic aox1 gene and the integrated efsha and efshb genes were approximately 2.2 kb, 891 bp and 969 bp respectively. These colonies were then selected and cultured in specific media for expression of the recombinant protein.

4.3. Western Analysis
    The presence of reFSH in each collected medium sample was confirmed by Western analysis. The Western blotting with specific polyclonal antibodies for each subunit revealed two bands of approximately 13 and 23 kDa, for eFSHa subunit, and two bands of approximately 18 and 29 kDa, corresponding to the eFSHb subunit (Figure 5). The bands began appearing 24 h after methanol induction, and strong bands were observed 54 h after methanol induction.

4.4. Immunoprecipitation    
    The IP analysis with a rabbit polyclonal antibody against the FSHb subunit (a common antibody that is used for both human and equine species) showed a similar pattern between the bands that appeared in the concentrated sample obtained 54 h after induction and the pure human FSH sample (positive control). As shown in Figure 6, two similar bands were found in each lane at the same position: the first band is approximately 29 kDa, and the second band is 50 kDa. We predicted that the smaller band corresponds to the FSHβ subunit as it could not be detected in the negative control, and the larger band can be corresponded either to the G-protein or our product.

5. Discussion
    The eFSH protein has been used to improve ovulation and embryo recovery rates, which can most likely increase the efficiency of the total embryo-transfer process (26). Jennings et al. (2009) reported that the stimulation of follicles with reFSH may be more potent than the stimulation with purified eFSH, which would lead to an increase in the follicular growth rate in mares (3). Thus, it is obvious that the production of reFSH has a practical application in the equine reproduction industry. Therefore, the current study provides the first report of the utilization of the yeast P. pastoris expression system for the secretion of a new variant of the Iranian Turkmen-thoroughbred horse fsh gene. Saneyoshi et al. (2001) reported a 462 bp sequence for efshb cDNA (27). The amino acid sequence that was deduced from their study is similar to the data that were reported by Fujiki et al. (1978) (3). However, our findings showed a 465 bp sequence for the efshb with some significant variations in 3΄-UTR. The heterologous expression of proteins with disulfide bonds often fails in bacteria (28, 29). As a eukaryotic expression system, P. pastoris was chosen because of its cost-effectiveness, potency for appropriate post translational modifications, secretion of foreign proteins into the culture medium (25), and a respiratory rather than a fermentative mode of growth that lowers the production of toxic waste materials in a high-cell-density environment (30). In fact, the expression of recombinant mammalian GTHs, such as porcine FSH (7), ovine FSH (3), and human chorionic gonadotropin (hCG) (31), in P. pastoris has shown equal biological activity compared with the native GTHs. Thus, P. pastoris was used for the expression of reFSH. We co-integrated the efshα and efshb genes into the P. pastoris genome by separated signal peptide sequences. This secretion pattern is consistent with the native form, in which the two subunits are co-expressed using two independent expression constructs. Yu et al. (2010) reported that the abovementioned form of protein secretion is much more efficient than the use of a common signal peptide leader (25). The integration of a foreign gene into the P. pastoris genome is a preferential method (3) that causes more stable transformants (32) and thus leads to a high expression level of the heterologous gene (3). The recombinant gene constructs were co-electroporated into the P. pastoris genome based on different sites to increase the possibility of the co-insertion of both gene constructs into the genome. The analysis of the Western blotting results revealed that each subunit of the reFSH molecule was successfully synthesized and secreted into the P. pastoris culture medium; however, the results showed two specific bands for each subunit. Based on reports by Kasuto and Levavi-Sivan (2005), it is likely that the larger protein bands (23 and 29 kDa bands in the Figure 5) are highly glycosylated forms of the subunits or multimers (33). The existence of some bands greater than 50 kDa that immunoreacted with the eFSHα and even the eFSHb antisera can be considered nonspecific bands that originated from the yeast (34). Although successful expression of recombinant FSH was reported in CHO by Fachal et al. (2010), the purification of this intracellular protein is often tedious and time consuming. In the current study, production of the recombinant eFSH was conducted in P. pastoris using extracellular secretion system. Application of this system resulted in secretion of the protein into the media which led to simple afterward purification. Post-translational modification is a concern in production of recombinant proteins in P. pastoris. However, this concern could be solved through application of Glyco-Switc strain with the ability of post translational modifications same as mammalian cells (35-37). Meanwhile, further studies are required to compare the functionality of the P. pastoris-derived eFSH in mares or other species.

Acknowledgments
    This study was substantially funded by the Royan Institute, Tehran, Iran. We express our appreciation to Gorgan University of Agricultural Sciences and Natural Resources and to the Royan proteomics lab for facilitating the research phases. In addition, the authors thank Zahra Mansouri and Najmeh Sadat Masoudi for their kind assistance.