ABSTRACT Recombinant chicken prolactin (chPRL), expressed in Escherichia coli and purified as a monomer, was successfully PEGylated and purified to homogeneity as a mono-PEGylated protein (PEG-chPRL). Its biological activity was estimated by its ability to interact with human prolactin receptor extracellular domain (hPRLR-ECD) and stimulate PRLR-mediated proliferation in Nb2-11C cells. PEG-chPRL activity in a cell bioassay was 10-fold lower than that of non-PEGylated chPRL, but only 2-fold lower in a binding assay to hPRLR-ECD. The CD spectra of non-PEGylated and PEGylated chPRL were almost identical and similar to that of hPRL, indicating proper refolding. Although the PEGylation of chPRL resulted in lower activity in vitro, PEG-chPRL was absorbed more slowly than chPRL, remained in the circulation 16 h longer. Furthermore the effects of PEG-chPRL injections in chickens on subsequent corticosteroid levels in blood were significantly profound compared to chPRL. These favorable PEGylation-induced pharmacokinetic alterations should improve efficacy of PEG-chPRL in in vivo experiments, as dosing frequency can be reduced due to its prolonged persistence in the circulation, and thus reduce the frequency of dosing. Furthermore, hydrophobic interaction chromatography was successfully adopted to isolate PEG-chPRL as a better alternative for separation of PEGylated PRL, and is likely to be successfully applicable to other proteins. INTRODUCTION Prolactin (PRL) is a polypeptide hormone that is mainly secreted by the pituitary gland and named for its ability to promote lactation (Scanes et al., 1975; Freeman et al., 2000). However, aside from its well-known lactogenic properties, over 300 different functions of PRL have been reported, highlighting the significance of this hormone. Prolactin is also synthesized by a number of extra pituitary tissues, including mammary gland, skin and immune cells (B- and T-lymphocytes, natural killers, and macrophages). This widespread expression partially explains its involvement in various processes, such as reproduction, immunomodulation, osmoregulation, growth, development, and metabolism in vertebrates (Freeman et al., 2000; Ben-Jonathan et al., 2008; Nguyen et al., 2008). In chickens, PRL plays an important role in the regulation of various physiological processes, such as induction and maintenance of incubation behavior (Sharp et al., 1979; Talbot et al., 1991; March et al., 1994), osmoregulation (Doneen and Smith, 1982; Murphy et al., 1986), immunomodulation (Bhat et al., 1983; Skwarlo-Sońta, 1990), and ovarian steroid secretion (Zadworny et al., 1989; Hrabia et al., 2004). Most of PRL’s diverse actions are mediated by binding of the non-modified 23-kDa PRL to its receptor (PRLR), a member of the class I cytokine receptor superfamily that contains a large extracellular domain (ECD), a transmembrane domain, and an intracellular domain (Tanaka et al., 1992; Bole-Feysot et al., 1998). This activates multiple intracellular signaling cascades, resulting in transcription of the genes that are needed for the tissue-specific changes induced by PRL (Brooks, 2012). Furthermore, a novel ligand of PRLR—prolactin-like protein (PRL-L; also called PRL2)—has been discovered in some non-mammalian vertebrate species, including chicken and zebrafish (Bu et al., 2015). Polyethylene glycol (PEG) has a long history of use as a non-toxic, non-immunogenic polymer, and PEGylated proteins are an important class of modern therapeutic drugs (Knop et al., 2010). One of the major benefits of PEGylation is an in vivo increase in half-life, primarily due to the formation of a stable covalent bond between activated PEG polymers and the peptides. Changes in the physical and chemical properties of the biological molecule result in improved solubility and stability, decreased immunogenicity, increased residence time of the conjugates in the blood, and reduced proteolysis and renal filtration rate, thereby allowing a lower dosing frequency (Kolate et al., 2014). However, heterologous PEGylation always results in the production of mixed PEGylated protein forms: mono, di, tri, and even higher. Thus, challenges in the synthesis of PEGylated proteins include low yield and difficulty in purifying the required (usually mono-PEGylated) product. To obtain the purified mono-PEGylated protein, the PEGylated proteins have to be separated from other reaction products, including non-reacted PEG, and then the mono-PEGylated protein has to be separated from it poly-PEGylated and non-PEGylated counterparts (Reichert and Borchard, 2016). The most commonly reported methods for purification are size-exclusion chromatography (SEC) and ion-exchange chromatography (Jevsevar et al., 2010). Even though PEGylation impacts protein hydrophobicity, hydrophobic interaction chromatography (HIC) has only been recently applied by us for the separation of mono-PEGylated human (h) PRL (Ocłoń et al., 2018). The present study has been undertaken with the aim to characterize the biological features of PEGylated form of chicken prolactin (chPRL) and to provide a novel tool for pharmacological homology studies in poultry research. Preliminary results of work were presented at the 46th Annual Meeting of The Israeli Endocrine Society (Ocłoń et al., 2017b). MATERIALS AND METHODS Materials Recombinant hPRL, rabbit PRL receptor extracellular domain (rbPRLR-ECD), and human prolactin receptor extracellular domain (hPRLR-ECD) were prepared in our laboratory as described previously (Gertler et al., 1992, 1996, 1998; Leibovich et al., 2001). Baf3 cells stably transfected with the long form of rbPRLR were obtained from Dr. Jean Djiane from Institut National de la Recherche Agronomique (INRA), Jouy-en-Josas Cedex, France; rat lymphoma cell line Nb2-11C was from Dr. Henri Friesen from Department of Physiology, University of Manitoba, Winnipeg, Canada, and Baf3/LP cells stably transfected with the long form of hPRLR were from Dr. Vincent Goffin, from Institute Necker Enfants Malades (INEM), Inserm U1151-CNRS UMR 8253, Paris Descartes University, France. Gibco RPMI-1640 medium and Gibco Dulbecco's modified Eagle's medium (DMEM) were from Invitrogen (Carlsbad, CA); fetal bovine serum and Pen-Strep solution (5 to 104 U/mL penicillin, 50 mg/mL streptomycin, 0.125 mg/mL fungisone) were from Biological Industries Ltd. (Beit Haemek, Israel); Superdex 75 HR 10/30 column and Q-Sepharose were from Pharmacia LKB Biotechnology AB (Uppsala, Sweden); NaCl, and Tris base were purchased from Bio-Lab Ltd. (Jerusalem, Israel). Glycerol, EDTA, HCl, Triton X-100, and urea were purchased from ENCO Diagnostics Ltd. (Petah-Tikva, Israel), and molecular markers for SDS-PAGE were purchased from Bio-Rad (Hercules, CA). Methoxy PEG propionylaldehyde – 20 kDa was purchased from Jenkem Technology Inc. (Allen, TX). Streptavidin-horseradish peroxidase conjugate (streptavidin–HRP) was purchased from Jackson ImmunoResearch (West Grove, PA) and 3,3΄,5,5΄-tetramethylbenzidine (TMB) was from Dako (DakoCytomation, Copenhagen, Denmark). ELISA kit for chPRL was from Aviva Systems Biology, Enco Scientific Service (Petah Tikva, Israel) and for chicken corticosterone (CORT) Eliza kit from Wuhan Fine Biotech Co., Ltd. (Wuhan, China). All reagents were of analytical or cell culture grade. Expression, Refolding, and Purification of ChPRL To improve the preparation of chPRL, synthetic cDNA encoding the chPRL sequence (NCBI Gene Database Accession Number NM_205466.2) was modified to ensure better codon usage and expression in Escherichia coli. The cDNA in pUC57 was digested with NcoI and HindIII, extracted, and ligated into linearized pMon3401 expression vector. E. coli MON105 competent cells were transformed with the new expression plasmid and plated on LB-agar plates containing 75 μg/mL spectinomycin for plasmid selection. Four E. coli colonies were isolated and confirmed to contain chPRL cDNA by digestion with NcoI/HindIII restriction enzymes. All colonies were positive and expressed chPRL upon stimulation with nalidixic acid (not shown). One colony was then sequenced to ensure the correct DNA sequence and used for large-scale fermentation in ten 2.5-L flasks, each containing 0.5 L Terrific Broth culture medium at 37°C. When the absorbance at 600 nm reached 0.9, nalidixic acid was added to 50 μg/mL. The bacteria were grown for an additional 24 h, centrifuged and frozen. Inclusion bodies were prepared and solubilized as described previously (Ocłoń et al., 2017a). Briefly, inclusion bodies prepared from 5 L of fermentation culture were solubilized in 20 mL of 50 mM Tris base, pH 8.5, containing 6 M guanidine-HCl and 8 mM 1.4-dithiothreitol. After 45 min of stirring at room temperature (RT), the solution was slowly added by peristaltic pump to 500 mL refolding buffer (50 mM Tris base, 160 mM arginine, 1 M urea, 4 mM cysteine, pH 8.5), stirred at 4°C for 24 h, and then the clear solution was dialyzed extensively against 10 mM Tris-HCl, pH 8 at 4°C and applied to a DEAE column (2.5 × 6 cm) pre-equilibrated with 10 mM Tris-HCl, pH 8. Elution was carried out using a discontinuous NaCl gradient in the same buffer (50, 100, 150, 300 mM NaCl). Fractions (40 mL) were collected and protein concentration was determined by absorbance at 280 and 260 nm. The monomer content of each fraction was determined by SEC on an analytical Superdex 75 HR 10/30 column. Fractions containing monomeric chPRL were pooled, dialyzed against NaHCO3 to ensure a 4:1 (w/w) protein-to-salt ratio, and lyophilized. Preparation of PEGylated ChPRL PEGylated chPRL was prepared according to the procedure of Ocłoń et al. (2018) with some modifications. Briefly, 50-mg aliquots of the purified chPRL were incubated with a 12-fold molar excess of methoxy PEG propionylaldehyde –20 kDa under conditions that favor PEGylation of the N-terminal amino group. ChPRL was dissolved in 1 M NaH2PO4 buffer, pH 6.5; then 425 μL of 1 M NaBH3CN was added, and the dissolved protein was conjugated with 5.22 mL PEG dissolved in 10 mM HCl. The reaction mixture was stirred for 24 h and dialyzed against 50 mM NaH2PO4, pH 6.5. The procedure was carried out at 4°C. Isolation of PEGylated ChPRL by HIC The chromatographic columns were packed with phenyl sepharose 6FF HS (20 mL bead volume) and pre-equilibrated with 50 mM NaH2PO4 containing 0.8 M ammonium sulfate, pH 6.5. Ammonium sulfate was added to the dialyzed solution to 0.8 M, and then applied at a flow rate of 0.8 mL/min to the column. The column was washed with 25 mL of 50 mM NaH2PO4 containing 0.8 M ammonium sulfate. The PEGylated protein was eluted by discontinuous gradient in 50 mM NaH2PO4, pH 6.5, containing 0.4, 0.2, or 0 M (NH4)2SO4. The eluate was monitored at 280 nm. Fractions containing the mono-PEGylated protein as determined by gel filtration on an analytical Superdex-200 column were pooled, dialyzed against NaHCO3, and lyophilized. Protein concentration was determined by absorbance at 280 nm using an extinction coefficient of 1.151 for a 0.1% solution of the protein part of the PEGylated chPRL (PEG-chPRL) protein. Determination of CD Spectra CD spectra were recorded using a JASCO J-810 spectropolarimeter in a 0.1-cm quartz cuvette for far-ultraviolet CD spectroscopy. Spectra were collected over 190 to 260 nm. Lyophilized chPRL and PEG-chPRL were dissolved in water, dialyzed against 50 mM phosphate buffer, pH 7.5 for 20 h, and adjusted to 50 μM concentration. The CD measurements were performed at 25°C as controlled by thermoelectric Peltier elements to an accuracy of 0.1°C. The CD spectra were measured in 5 repetitions, resulting in an average spectrum for each protein. Standard deviation of the average CD signal at 222 nm was in the 5% range. For secondary structure determination, the CD data were expressed in degree × cm2/dmol per mean residue, based on respective molecular mass. For secondary structure analysis, the DICHROWEB program (Whitmore and Wallace, 2004) was used and the α-helical content was calculated using the analytical program SELCON3 (Sreerama and Woody, 2000). Determination of Purity and Monomer Content SDS-PAGE was carried out according to Laemmli (1970) in a 15% polyacrylamide gel under reducing and non-reducing conditions. The gel was stained with Coomassie Brilliant Blue R. SEC was performed on a Superdex 75 HR 10/30 column for determination of rbPRLR-ECD and chPRL and on a Superdex 200 HR 10/30 column for PEG-chPRL. The column was pre-equilibrated and developed using 25 mM Tris-HCl buffer, pH 8.0 containing 300 mM NaCl (TN buffer) at RT. The columns were calibrated with leptin (16 kDa), human growth hormone (21.5 kDa), albumin (66 kDa), and bovine IgG monomer (150 kDa) and dimer (300 kDa). Determination of Complex Stoichiometry To characterize the binding stoichiometry between chPRL, or PEG-chPRL and rbPRLR-ECD, they were mixed in different molar ratios, incubated for 30 min at 4°C, and then separated under non-denaturing conditions by SEC using an analytical Superdex 75 HR 10/30 column equilibrated with TN buffer (pH 9) at RT as described previously (Niv-Spector et al., 2005). Binding Assay A binding assay was performed using rbPRLR-ECD as the receptor source. In-house-prepared biotinylated hPRL (Ocłoń et al., 2018) served as the ligand that could be competed off by the respective PEGylated or non-PEGylated chPRL. Polystyrene 96-well microtiter plates were coated overnight at 4°C with 100 μL of 31 μM rbPRLR-ECD in PBS, pH 7.4. Wells were then washed once with PBST (PBS containing 0.05% w/v Tween 80) and blocked with PBS containing 3% (w/v) skim milk for 2 h at RT. All further incubations were also carried out at RT. Wells were washed again once with PBST and incubated with different concentrations of unlabeled PRL (50 μL/well, in 3 replicates) for 30 min, and then 50 μL of biotinylated hPRL was added to each well for another 1.5 h. Then the wells were washed 3 times with PBST and incubated with 1:10,000 streptavidin–HRP in PBST for 1 h. Wells were washed 3 times with PBST, and the reaction was quantified at 450 nm by ELISA Micro-Plate Reader ELx808, BioTek Instruments Inc. (Winooski, VT) using TMB according to the manufacturer's instructions. Surface Plasmon Resonance Surface plasmon resonance was performed on a BIAcore T200 instrument (Uppsala, Sweden). PEGylated or non-PEGylated chPRL was diluted in 100 mM Na-acetate (pH 4.6) to 20 μg/mL and immobilized on a CM5 sensor chip. The protein solution was then run over the chip for 5 min at a rate of 10 mL/min. The binding assay was performed by injecting the analyte (hPRLR-ECD) solution at 6 different concentrations (10, 14, 20, 25, 33, 50 nM) at a flow rate of 20 μL/min at 25°C. These conditions resulted in a linear relationship between protein concentration and maximal (steady-state) response, indicating a pseudo first-order regime in relation to the immobilized ligand. The net signal was obtained by subtracting the blank signal (dextran matrix). The association phase for analyte binding to both ligands was followed for 4 min, and dissociation phases were monitored for 3 min. The response was monitored as a function of time (sensorgram) at 25°C. Data were fit using BIAevaluation 3.2 software. Nb2-11C Proliferation Assay The proliferation rate of Nb2-11C cells (expressing the short form of rat PRLR) was used to estimate the biological activity of PEGylated and non-PEGylated chPRL as described previously (Ocłoń et al., 2017a). Briefly, Nb2-11C cells were maintained in RPMI-1640 medium containing 5% (v/v) fetal calf serum supplemented with antibiotic–antimycotic solution (10,000 units penicillin, 10 mg streptomycin, and 25 μg amphotericin B per mL). A day before the assay, cells were transferred to 5% horse (gelding) serum medium overnight. The cells were then plated in 96-well plates at 100 μL (2.5 × 104 cell/well) with different concentrations of PRL (10 μL of 50, 12.5, 3.125, 0.781, 0.195, and 0.045 μg/mL for non-PEGylated chPRL, or 10-fold higher concentrations of PEGylated chPRL) and incubated under a humidified atmosphere of 95% air and 5% CO2 at 37°C. Cell number was assessed 48 h after PRL induction by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide assay. Growth curves were drawn using the Prizm (4.0) (Prisma, GraphPad Prism version 4.0, GraphPAD Software, San Diego, CA) non-linear regression sigmoidal dose-response curve and the EC50 values were calculated. Comparative Pharmacokinetics of PEGylated and Non-PEGylated ChPRL in Broilers All of the procedures in this study were carried out in accordance with the accepted ethical and welfare standards of the Israel Ethics Committee (IL-711/17). Cobb 500 strain broiler chickens (Gallus domesticus), 16 d of age and weighing ∼0.5 kg, were used for all pharmacokinetic studies. Each chick was individually tagged and weighed. The chicks were divided into 2 groups, 3 chicks each, which were kept together in battery cages. Water and feed in mash form were available for ad libitum consumption. The diet was designed to meet or exceed NRC (1994) recommendations. The bird groups were administered a single intraperitoneal injection of chPRL (3 mg/kg) or PEG-chPRL (3 mg/kg), using a 25-gauge needle with a 0.4 mm bore and 25 mm length. The chicks’ blood was collected from the jugular vein into a heparinized 2.0-mL syringe via a 23-gauge needle for each time point (before injection and after 0.5, 2, 4, and 20 h). The samples were centrifuged, and the plasma was harvested and stored at –80°C until the assay. Plasma chPRL and PEG-chPRL concentrations were measured using a commercial ELISA kit according to the manufacturer's protocol. As the interaction of PEGylated chPRL is likely not identical to that of non-chPEGylated PRL, the former's concentration was determined according to the standard curve of PEG-chPRL. Briefly, a 96-well plate was pre-coated with anti-chPRL antibody. Standards and test samples were added to the wells, incubated with biotinylated detector antibody specific for PRL for 60 min and then washed. Avidin-HRP conjugate was then added, incubated for 45 min and unbound conjugate was washed away. An enzymatic reaction was produced through the addition of TMB substrate which was catalyzed by HRP, generating a blue-colored product that changed to yellow after adding acidic stop solutions. Comparative In Vivo Effect of PEGylated and Non-PEGylated ChPRL on CORT Plasma Concentrations in Chickens The animal experiment was conducted according to research protocol approved by the II Local Animal Ethics Committee headquartered at the Institute of Pharmacology Polish Academy of Sciences in Krakow, Poland (approval no. 54/2017). Laying Hy-Line Brown hens at 27 weeks of age were kept in individual cages at a neutral temperature and under a photoperiodic regime of 14L:10D and fed ad libitum with commercial food (11.5 MJ/kg metabolizable energy, 17.5% proteins) and water. Birds were divided into 4 groups (n = 7 chickens each) and injected subcutaneously, daily through the whole period with 250 (group I) or 500 (group II) μg of recombinant chPRL, or 125 (group III) or 250 μg of recombinant PEG-chPRL (group IV) in 0.2 mL/kg BW diluted in 0.9% NaCl. The doses of chPRL were chosen according to previous in vivo study (Rozenboim et al., 1993), and our preliminary experiment (AH, unpublished data), taking into account the concentrations of PRL in blood plasma. Before hormone injection (day 0) and on days 3 and 9 of the experiment, about 2 h after oviposition, and 24 h after last chPRL or last PEG-chPRL injection, blood was taken from the wing vein of hens into heparinized tubes. Plasma was collected after centrifugation (3,000× g, 5 min) and kept at –20°C until CORT determinations. Plasma chicken CORT concentrations were measured using a commercial Competitive ELISA kit according to the manufacturer's protocol (intra assay CV < 8%, inter assay CV < 10%). The microtiter plate was pre-coated with chicken CORT. During the reaction, CORT in the sample competed with a fixed amount of CORT on solid phase supporter for sites on the Biotinylated Detection Antibody specific to CORT during 45 min incubation at 37°C. Excess conjugate and unbound sample was washed from the plate, and HRP-Streptavidin was added and incubated for 30 min at 37°C. Then TMB substrate solution was added to each well and incubated in dark within 15 min at 37°C. The enzyme-substrate reaction was terminated by the addition of sulfuric acid solution and the color change was measured spectrophotometrically at a wavelength of 450 nm, using an ELISA plate reader Biotek EPOCH2 (Winooski, USA). The results were analyzed for statistically significant differences by 2-way ANOVA (effect of dose × PRL), followed by Duncan's test. RESULTS Purification and Physicochemical Characterization of Non-PEGylated and PEGylated ChPRL The refolded and dialyzed chPRL was purified on a DEAE anion-exchange column equilibrated with 10 mM Tris base buffer pH 8 using a non-continuous NaCl gradient (see Materials and Methods). The monomeric fractions of chPRL, as documented by analytical SEC analysis, were eluted with 100 mM NaCl. The isolated chPRL was over 95% pure as shown by SDS-PAGE carried out under reducing conditions (Figure 1, lane 2) and consisted of 95% monomers with a small amount of dimers preceding the main peak, as documented by SEC analysis (Figure 2A). The average yield from 2 preparations was 150 to 180 mg of pure chPRL from 5 L of fermentation culture. PEGylation of this protein, carried out as described in Materials and Methods, yielded ∼12 mg of PEG-chPRL from 50 mg of non-PEGylated PRL. The purity and homogeneity of the PEG-chPRL were also documented by SDS-PAGE (Figure 1, lane 1), with the main band appearing at ∼ 55 kDa. The gel was intentionally overloaded to detect small amounts of accompanying proteins. Small amounts of double-PEGylated (molecular mass of 90 kDa) and non-PEGylated chPRL were also detected. The PEGylation reaction monitored on a Superdex 200 HR 10/30 column exhibited 90% mono-PEGylated chPRL (Figure 2D). The calculated molecular mass of this peak, however, was ∼220 kDa, in contrast to the theoretical value of 43 kDa, likely due to increased hydrodynamic volume. Figure 1. View largeDownload slide SDS-PAGE analysis of PEG-chPRL (15 μg, lane 1), chPRL (10 μg, lane 2) on a 15% gel in the presence of reducing agent. Colored molecular mass markers (lane 3, from top to bottom in kDa: 100, 75, 63, 48, 35, 25, 20, 17, and 11) usually run slower than the corresponding non-colored proteins. Figure 1. View largeDownload slide SDS-PAGE analysis of PEG-chPRL (15 μg, lane 1), chPRL (10 μg, lane 2) on a 15% gel in the presence of reducing agent. Colored molecular mass markers (lane 3, from top to bottom in kDa: 100, 75, 63, 48, 35, 25, 20, 17, and 11) usually run slower than the corresponding non-colored proteins. Figure 2. View largeDownload slide SEC of the purified proteins and their complexes with rbPRLR-ECD. (A) ChPRL, (B) rbPRLR-ECD, (C) chPRL–rbPRLR-ECD complex, (D) PEG-chPRL, (E) rbPRLR-ECD, (F) PEG-chPRL–rbPRLR-ECD complex. (A–C) Developed on a Superdex HR 75 10/30 column in TN buffer pH 8 at 0.8 mL/min. (D–F) Developed on a Superdex HR 200 10/30 column in TN buffer pH 8 at 0.7 mL/min. The separation was carried out at room temperature and the proteins were detected at 280 nm. Figure 2. View largeDownload slide SEC of the purified proteins and their complexes with rbPRLR-ECD. (A) ChPRL, (B) rbPRLR-ECD, (C) chPRL–rbPRLR-ECD complex, (D) PEG-chPRL, (E) rbPRLR-ECD, (F) PEG-chPRL–rbPRLR-ECD complex. (A–C) Developed on a Superdex HR 75 10/30 column in TN buffer pH 8 at 0.8 mL/min. (D–F) Developed on a Superdex HR 200 10/30 column in TN buffer pH 8 at 0.7 mL/min. The separation was carried out at room temperature and the proteins were detected at 280 nm. To characterize the folding propensity of PEGylated as compared to non-PEGylated chPRL, CD analysis was performed at pH 7.5 (Figure 3). The respective α-helical content values of chPRL and PEG-chPRL were 43% and 49%, and the corresponding β-sheet contents were 8% and 9%, characteristic of all known PRL, indicating proper refolding. Figure 3. View largeDownload slide Secondary structure of recombinant PEG-chPRL and chPRL. The CD spectra of purified recombinant chPRL and PEG-chPRL were collected over 190 to 260 nm at 25°C. Lyophilized chPRL and PEG-chPRL were dissolved in water, dialyzed against 50 mM phosphate buffer, pH 7.5, for 20 h, and adjusted to 50 μM. For secondary structure analysis, the DICHROWEB program was used and the α-helical content was calculated using the analysis program SELCON3 Figure 3. View largeDownload slide Secondary structure of recombinant PEG-chPRL and chPRL. The CD spectra of purified recombinant chPRL and PEG-chPRL were collected over 190 to 260 nm at 25°C. Lyophilized chPRL and PEG-chPRL were dissolved in water, dialyzed against 50 mM phosphate buffer, pH 7.5, for 20 h, and adjusted to 50 μM. For secondary structure analysis, the DICHROWEB program was used and the α-helical content was calculated using the analysis program SELCON3 Detection of ChPRL and PEG-ChPRL Complex Formation with RbPRLR-ECD To characterize the binding stoichiometry between non-PEGylated or PEGylated chPRL and rbPRLR-ECD, the experiment was performed using either a constant 15 μM PEG-chPRL or chPRL and increasing concentrations of rbPRLR-ECD or vice versa. SEC analysis using analytical Superdex 200 (for PEG-chPRL) or Superdex HR 75 10/30 (for chPRL) columns was performed to determine the molecular mass of the binding complex under non-denaturing conditions. All components added alone were eluted from the column as monomers at RT of 15.34 min for chPRL (Figure 2A) or 15.44 min for PEG-chPRL (Figure 2D). The RT of rbPRLR-ECD on Superdex 75 and Superdex 200 columns were, respectively, 15.35 and 23.21 min (Figure 2B and E, respectively). Mixing chPRL and rbPRLR-ECD at a 1:1 molar ratio resulted in a single peak with an RT of 13.95 min (Figure 2C). The molecular mass of this peak as calculated by calibration with different proteins was 45 kDa, close to the expected theoretical value of 43 kDa. Though the RT for the PEG-chPRL–rbPRLR-ECD complex (15.31 min) was only slightly higher than that of PEG-chPRL (15.44 min), the peak of rbPRLR-ECD (23.27 min) almost completely disappeared, documenting formation of the 1:1 PEG-chPRL–rbPRLR-ECD complex (Figure 2F). Binding Experiments and Biological Activity Assay The binding activities of non-PEGylated and PEGylated chPRL were analyzed by 2 assays. In the competitive non-radioactive binding assay (Figure 4A) using biotinylated hPRL as the ligand and hPRLR-ECD as the receptor, PEGylation lowered the affinity of PEG-chPRL 1.8-fold (EC50 = 3.27 × 10−5 M) compared to non-PEGylated chPRL (EC = 1.82 × 10−5 M). To better characterize the binding affinities of non-PEGylated and PEGylated chPRL with hPRLR-ECD, surface plasmon resonance methodology was also used. The respective kinetic constants (kon) for chPRL and PEG-chPRL were 11.1 and 11.7 m/s, whereas the respective koff constants were 9.10 × 10−3 s−1 and 7.44 × 10−3 s−1, and the calculated thermodynamic association constants (Ka) were 1.25 × 103 and 1.57 × 103 m, respectively, showing no difference. Figure 4. View largeDownload slide Binding and bioassay experiments. (A) Competitive displacement of biotinylated hPRL bound to plated rbPRLR-ECD by chPRL or PEG-chPRL. (B) Comparison of biological activity of PEGylated and non-PEGylated chPRL in Nb2-11C cells. For other details, see text. Figure 4. View largeDownload slide Binding and bioassay experiments. (A) Competitive displacement of biotinylated hPRL bound to plated rbPRLR-ECD by chPRL or PEG-chPRL. (B) Comparison of biological activity of PEGylated and non-PEGylated chPRL in Nb2-11C cells. For other details, see text. The biological activity of non-PEGylated and PEGylated chPRL was tested in Nb2-11C cells using cell-proliferation rate as the readout. Unlike the binding which showed little or no difference, PEGylation of chPRL reduced its biological activity approximately 10-fold (Figure 4B). Pharmacokinetic Studies A blood sample taken prior to injection showed 1.41 ± 0.25 ng/mL (mean ± SD, n = 3). As shown in Figure 5, after 30 min, the levels of both PEGylated and non-PEGylated chPRL were 300 to 400 ng/mL. The level of non-PEGylated chPRL dropped rapidly to 30 and 1.4 ng/mL after 2 and 4 h and to less than 1 ng/mL after 20 h. In contrast, the decline of PEGylated chPRL was much slower, reaching 109, 50, and 38 ng/mL, respectively. Figure 5. View largeDownload slide Plasma levels of non-PEGylated chPRL (filled squares), and PEG-chPRL (open squares) after a single intraperitoneal injection of 3 mg protein/kg (n = 3). Plasma levels of the proteins after 30 min, 2, 4 and 20 h postinjection were measured by ELISA. Figure 5. View largeDownload slide Plasma levels of non-PEGylated chPRL (filled squares), and PEG-chPRL (open squares) after a single intraperitoneal injection of 3 mg protein/kg (n = 3). Plasma levels of the proteins after 30 min, 2, 4 and 20 h postinjection were measured by ELISA. The half-lives in blood as calculated by Prizma program between 0.5 and 20 h were respectively 41 and 435 min for non-PEGylated and PEGylated chPRL. Surprisingly, the concentration of PEGylated chPRL changed only slightly between 4 and 20 h from 49.4 ± 7.9 to 37.0 ± 6.7 ng/mL (mean ± SEM). It is not clear if this is an experimental error or an indication of another concentration-dependent clearance mechanism. In Vivo Effect of ChPRL and PEG-chPRL To compare the in vivo effects of chPRL and PEG-chPRRL on CORT plasma concentrations, known as a PRL-responsive readout (Skwarło-Sonta et al.,1987; Scanes, 2016) were used. The results presented in Table 1 clearly show that the effect achieved by PEG-chPRL 24 h after the last injection was significantly higher compared to that of chPRL. Such effect was even seen despite the fact that the concentration of the injected PEG-chPRL was 4-fold lower than that of non-pegylated chPRL. Table 1. Effect of chPRL and PEG-chPRL injections on corticosterone levels (ng/mL, mean ± SEM) in blood, determined 24 h after the corresponding injection. Day Ch-PRL (250 μg/kg BW/d) Ch-PRL (500 μg/kg BW/d) PEG-chPRL (125 μg/kg BW/d) PEG-chPRL (250 μg/kg BW) 0 26.7 ± 2.8a (18)* 3 46.7 ± 2.5b (5) 40.3 ± 2.1b (4) 56.3 ± 2.5c (5) 86.8 ± 3.6d (5) 9 51.6 ± 2.5b (5) 48.9 ± 4.8b,c (5) 72.3 ± 5.1d (4) 80.9 ± 3.0d (5) Day Ch-PRL (250 μg/kg BW/d) Ch-PRL (500 μg/kg BW/d) PEG-chPRL (125 μg/kg BW/d) PEG-chPRL (250 μg/kg BW) 0 26.7 ± 2.8a (18)* 3 46.7 ± 2.5b (5) 40.3 ± 2.1b (4) 56.3 ± 2.5c (5) 86.8 ± 3.6d (5) 9 51.6 ± 2.5b (5) 48.9 ± 4.8b,c (5) 72.3 ± 5.1d (4) 80.9 ± 3.0d (5) *The numbers in parentheses indicate the number of animals. Means designated with different letters are significantly different (P < 0.05). View Large Table 1. Effect of chPRL and PEG-chPRL injections on corticosterone levels (ng/mL, mean ± SEM) in blood, determined 24 h after the corresponding injection. Day Ch-PRL (250 μg/kg BW/d) Ch-PRL (500 μg/kg BW/d) PEG-chPRL (125 μg/kg BW/d) PEG-chPRL (250 μg/kg BW) 0 26.7 ± 2.8a (18)* 3 46.7 ± 2.5b (5) 40.3 ± 2.1b (4) 56.3 ± 2.5c (5) 86.8 ± 3.6d (5) 9 51.6 ± 2.5b (5) 48.9 ± 4.8b,c (5) 72.3 ± 5.1d (4) 80.9 ± 3.0d (5) Day Ch-PRL (250 μg/kg BW/d) Ch-PRL (500 μg/kg BW/d) PEG-chPRL (125 μg/kg BW/d) PEG-chPRL (250 μg/kg BW) 0 26.7 ± 2.8a (18)* 3 46.7 ± 2.5b (5) 40.3 ± 2.1b (4) 56.3 ± 2.5c (5) 86.8 ± 3.6d (5) 9 51.6 ± 2.5b (5) 48.9 ± 4.8b,c (5) 72.3 ± 5.1d (4) 80.9 ± 3.0d (5) *The numbers in parentheses indicate the number of animals. Means designated with different letters are significantly different (P < 0.05). View Large DISCUSSION Using a relatively simple protocol, we produced 150 to 180 mg of pure recombinant chPRL from 5 L of bacterial culture. PEGylation of this protein resulted in a yield ∼12 mg of PEG-chPRL from 50 mg of non-PEGylated chPRL. Results of the CD analysis of PEGylated and non-PEGylated chPRL were similar, indicating that PEGylation does not affect the secondary structure, similar to hPRL (Ocłoń et al., 2018), suggesting proper refolding. PEGylation of chPRL yielded mainly mono-PEGylated species with a slight, if any, reduction in affinity for rbPRLR-ECD. To purify PEG-chPRL, the HIC purification method was successfully adopted. Use of these conditions resulted in purity of mono-PEGylated chPRL comparable to that obtained using SEC. Thus, HIC represents a viable alternative for separation of PEGylated PRLs, which can be successfully applied to other proteins. Furthermore, the biological activities tested in the Nb2-11C assay showed 10-fold lower activity of PEGylated chPRL. As PRL is well-documented to have 2 binding sites and activate its receptor by dimerization or conformational change of the predimerized receptor (Waters and Brooks, 2015), the lower biological activity can be attributed to the fact that PEG-chPRL binds well to the isolated rbPRLR-ECD through its binding site 1, but probably lesser through binding site 2. Although the PEGylation of chPRL reduced its activity in vitro, consistent with the observations of other cytokines, such as leptin (Elinav et al., 2009), interleukin 22 (Niv-Spector et al., 2012), and hPRL (Ocłoń et al., 2018), a more favorable pharmacokinetic profile could compensate for its reduced in vitro potency as shown for mouse leptin antagonist. Thus, we examined the pharmacokinetics of both chPRL and PEG-chPRL in chicks, and showed that PEG-chPRL remains in the circulation much longer. These favorable pharmacokinetic alterations afforded by PEGylation should improve the efficacy of PEG-chPRL, and possibly reduce the frequency of dosing in in vivo experiments. We decided to estimate the biological activity of PEG-chPRL by measuring the CORT plasma concentration in laying chickens. As previously has been shown, PRL can promote CORT secretion (Miller et al., 2009), and PRL injection into chick embryos dramatically raises plasma CORT within 2 h (Kühn et al., 1996). Furthermore, it has also been revealed in in vitro study that PRL limited CORT degradation to 5α-reduced metabolites by an acute inhibition of the activity of 5α-reductase (Carsia et al., 1984), which is known to degrade of CORT to 5α-dihydrocorticosterone and 5α-tetrahydrocorticosterone. This acute action of PRL is Ca2+-dependent and occurs with a half-effective concentration (EC50) of 50 ng/ml, a concentration that is well within the physiological range of circulating PRL (Carsia et al., 1987). In our study, both PRLs increased plasma CORT levels; however, the biological activity of PEG-chPRL was significantly higher than that of non-pegylated chPRL, which seems to confirm the usefulness of PEG-chPRL as a tool for long-term avian physiological studies. ACKNOWLEDGMENTS This research was partially supported by the grant DS-3243/KFiEZ. REFERENCES Ben-Jonathan N. , LaPensee C. R. , LaPensee E. W. . 2008 . What can we learn from rodents about prolactin in humans? Endocr. Rev. 29 : 1 – 41 . Google Scholar CrossRef Search ADS PubMed Bhat G. , Gupta S. K. , Maiti B. R. . 1983 . Influence of prolactin on mitotic activity of the bursa of Fabricius of the chick . Gen. Comp. Endocrinol. 52 : 452 – 455 . Google Scholar CrossRef Search ADS PubMed Bole-Feysot C. , Goffin V. , Edery M. , Binart N. , Kelly P. A. . 1998 . Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice . Endocr. Rev. 19 : 225 – 268 . Google Scholar CrossRef Search ADS PubMed Brooks C. L. 2012 . Molecular mechanisms of prolactin and its receptor . Endocr. Rev. 33 : 504 – 525 . Google Scholar CrossRef Search ADS PubMed Bu G. , Liang X. , Li J. , Wang Y. . 2015 . Extra-pituitary prolactin (PRL) and prolactin-like protein (PRL-L) in chickens and zebrafish . Gen. Comp. Endocrinol. 220 : 143 – 153 . Google Scholar CrossRef Search ADS PubMed Carsia R. V. , Scanes C. G. , Malamed S. . 1984 . Self-suppression of corticosteroidogenesis: evidence for a role of adrenal 5-α reductase . Endocrinol . 115 : 2464 – 2472 . Google Scholar CrossRef Search ADS Carsia R. V. , Scanes C. G. , Malamed S. . 1987 . Polyhormonal regulation of avian and mammalian corticosteroidogenesis in vitro . Comp. Biochem. Physiol. 88 : 131 – 140 . Google Scholar CrossRef Search ADS Doneen B. A. , Smith T. E. . 1982 . Ontogeny of endocrine control of osmoregulation in chick embryo . Gen. Comp. Endocrinol . 48 : 310 – 318 . Google Scholar CrossRef Search ADS PubMed Elinav E. , Niv-Spector L. , Katz M. , Price T. O , Ali M. , Yacobovitz M. , Solomon G , Reicher S. , Lynch J. L , Halpern Z. , Banks W. A. , Gertler A. . 2009 . Pegylated leptin antagonist is a potent orexigenic agent: preparation and mechanism of activity . Endocrinology 150 : 3083 – 3091 . Google Scholar CrossRef Search ADS PubMed Freeman M. E. , Kanyicska B. , Lerant A. , Nagy G. . 2000 . Prolactin: structure, function, and regulation of secretion . Physiol. Rev . 80 : 1523 – 1631 . Google Scholar CrossRef Search ADS PubMed Gertler A. , Grosclaude J. , Strasburger C. J. , Nir S. , Djiane J. . 1996 . Real-time kinetic measurements of the interactions between lactogenic hormones and prolactin-receptor extracellular domains from several species support the model of hormone-induced transient receptor dimerization . J. Biol. Chem. 271 : 24482 – 24491 . Google Scholar CrossRef Search ADS PubMed Gertler A. , Hauser S. D. , Sakal E. , Vashdi D. , Staten N. , Freeman J. J. , Krivi G. G. . 1992 . Preparation, purification, and determination of the biological activities of 12 N terminus-truncated recombinant analogues of bovine placental lactogen . J. Biol. Chem . 267 : 12655 – 12659 . Google Scholar PubMed Gertler A. , Simmons J. , Keisler D. H. . 1998 . Large-scale preparation of biologically active recombinant ovine obese protein (leptin) . FEBS Lett. 422 : 137 – 140 . Google Scholar CrossRef Search ADS PubMed Hrabia A. , Paczoska-Eliasiewicz H. , Rzasa J. . 2004 . Effect of prolactin on estradiol and progesterone secretion by isolated chicken ovarian follicles . Folia Biol. (Krakow) 52 : 197 – 203 . Google Scholar CrossRef Search ADS PubMed Jevsevar S. , Kunstelj M. , Porekar V. G. . 2010 . PEGylation of therapeutic proteins . Biotechnol. J. 5 : 113 – 128 . Google Scholar CrossRef Search ADS PubMed Knop K. , Hoogenboom R. , Fischer D. , Schubert U. S. . 2010 . Poly(ethylene glycol) in drug delivery: pros and cons as well as potential alternatives . Angew. Chem. Int. Ed. 49 : 6288 – 6308 . Google Scholar CrossRef Search ADS Kolate A. , Baradia D. , Patil S. , Vhora I. , Kore G. , Misra A. . 2014 . PEG – a versatile conjugating ligand for drugs and drug delivery systems . J. Control. Release 192 : 67 – 81 . Google Scholar CrossRef Search ADS PubMed Kühn E. R. , Shimada K. , Ohkubo T. , Vleurick L. M. , Berghman L. R. , Darras V. M. . 1996 . Influence of recombinant chicken prolactin on thyroid hormone metabolism in the chick embryo . Gen. Comp. Endocrinol. 103 : 349 – 358 . Google Scholar CrossRef Search ADS PubMed Laemmli U. K. 1970 . Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227 : 680 – 685 . Google Scholar CrossRef Search ADS PubMed Leibovich H. , Raver N. , Herman A. , Gregoraszczuk E. L. , Gootwine E. , Gertler A. . 2001 . Large-scale preparation of recombinant ovine prolactin and determination of its in vitro and in vivo activity . Protein Expr. Purif. 22 : 489 – 496 . Google Scholar CrossRef Search ADS PubMed March B. , Sharp P. J. , Wilson P. W. , Sang H. M. . 1994 . Effect of active immunization against recombinant-derived chicken prolactin fusion protein on the onset of broodiness and photoinduced egg laying in bantam hens . Reproduction 101 : 227 – 233 . Google Scholar CrossRef Search ADS Miller D. A. , Vleck C. M. , Otis D. L. . 2009 . Individual variation in baseline and stress-induced corticosterone and prolactin levels predicts parental effort by nesting mourning doves . Horm. Behav. 56 : 457 – 464 . Google Scholar CrossRef Search ADS PubMed Murphy M. J. , Brown P. S. , Brown S. C. . 1986 . Osmoregulatory effects of prolactin and growth hormone in embryonic chicks . Gen. Comp. Endocrinol. 62 : 485 – 492 . Google Scholar CrossRef Search ADS PubMed Nguyen N. , Stellwag E. J. , Zhu Y. . 2008 . Prolactin-dependent modulation of organogenesis in the vertebrate: Recent discoveries in zebrafish . Comp. Biochem. Physiol. Part C Toxicol. Pharmacol 148 : 370 – 380 . Google Scholar CrossRef Search ADS Niv-Spector L. , Gonen-Berger D. , Gourdou I. , Biener E. , Gussakovsky E. E. , Benomar Y. , Ramanujan K. V. , Taouis M. , Herman B. , Callebaut I. , Djiane J. , Gertler A. . 2005 . Identification of the hydrophobic strand in the A B loop of leptin as major binding site III: implications for large-scale preparation of potent recombinant human and ovine leptin antagonists . Biochem. J. 391 : 221 – 230 . Google Scholar CrossRef Search ADS PubMed Niv-Spector L. , Shpilman M. , Levi-Bober M. , Katz M. , Varol C. , Elinav E. , Gertler A. . 2012 . Preparation and characterization of mouse IL-22 and its four single-amino-acid muteins that act as IL-22 receptor-1 antagonists . Protein Eng. Des. Sel . 25 : 397 – 404 . Google Scholar CrossRef Search ADS PubMed NRC . 1994 . Nutrient requirements of poultry . Ninth Rev ed . National Academy Press , Washington D.C . Ocłoń E. , Leśniak-Walentyn A. , Solomon G. , Shpilman M. , Gertler A. . 2017a . Comparison of in vitro bioactivity of chicken prolactin and mammalian lactogenic hormones . Gen. Comp. Endocrinol. 240 : 27 – 34 . Google Scholar CrossRef Search ADS Ocłoń E. , Solomon G. , Hayouka Z. , Salame T. M. , Goffin V. , Gertler A. . 2017a . Novel reagents for human prolactin research: large-scale preparation and characterization of prolactin receptor extracellular domain, non-pegylated and pegylated prolactin and prolactin receptor antagonist . Protein Eng. Des. Sel . 31 : 7 – 16 . Google Scholar CrossRef Search ADS Ocłoń E. , Solomon G. , Gertler A. . 2017b . Preparation, characterization and in vitro evaluation of non-pegylated and pegylated chicken and human prolactins . Page 123 . 46th Annual Meeting of the Israeli Endocrine Society. Abstract Book. Tel Aviv, Israel . Reichert C. , Borchard G. . 2016 . Noncovalent PEGylation, an innovative subchapter in the field of protein modification . J. Pharm. Sci. . 105 : 386 – 390 . Google Scholar CrossRef Search ADS PubMed Rozenboim I. , Tabibzadeh C. , Silsby J. L , El Halawani M. E . 1993 . Effect of ovine prolactin administration on hypothalamic vasoactive intestinal peptide (VIP), gonadotropin releasing hormone I and II content, and anterior pituitary VIP receptors in laying turkey hens . Biol. Reprod . 48 : 1246 – 1250 . Google Scholar CrossRef Search ADS PubMed Scanes C. G. 2016 . Biology of stress in poultry with emphasis on glucocorticoids and the heterophil to lymphocyte ratio . Poult. Sci. 95 : 2208 – 2215 . Google Scholar CrossRef Search ADS PubMed Scanes C. G. , Bolton N. J , Chadwick A. . 1975 . Purification and properties of an avian prolactin . Gen. Comp. Endocrinol. 27 : 371 – 379 . Google Scholar CrossRef Search ADS PubMed Sharp P. J. , Scanes C. G. , Williams J. B. , Harvey S. , Chadwick A. . 1979 . Variations in concentrations of prolactin, luteinizing hormone, growth hormone and progesterone in the plasma of broody bantams (Gallus domesticus) . J. Endocrinol. . 80 : 51 – 57 . Google Scholar CrossRef Search ADS PubMed Skwarło-Sońta K. 1990 . Mitogenic effect of prolactin on chicken lymphocytes in vitro . Immunol. Lett. 24 : 171 – 177 . Google Scholar CrossRef Search ADS PubMed Skwarlo-Sonta K. , Sotowska-Brochocka J. , Rosolowska-Huszcz D. , Pawlowska-Wojewódka E. , Gajewska A. , Stepień D. , Kochan K. . 1987 . Effect of prolactin on the diurnal changes in immune parameters and plasma corticosterone in white leghorn chickens . Acta Endocrinol. (Copenh). 116 : 172 – 178 . Google Scholar PubMed Sreerama N. , Woody R. W. . 2000 . Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set . Anal. Biochem. 287 : 252 – 260 . Google Scholar CrossRef Search ADS PubMed Talbot R. T. , Hanks M. C. , Sterling R. J. , Sang H. M. , Sharp P. J. . 1991 . Pituitary prolactin messenger ribonucleic acid levels in incubating and laying hens: effects of manipulating plasma levels of vasoactive intestinal polypeptide . Endocrinology 129 : 496 – 502 . Google Scholar CrossRef Search ADS PubMed Tanaka M. , Maeda K. , Okubo T. , Nakashima K. . 1992 . Double antenna structure of chicken prolactin receptor deduced from the cDNA sequence . Biochem. Biophys. Res. Commun. 188 : 490 – 496 . Google Scholar CrossRef Search ADS PubMed Waters M. J. , Brooks A. J. . 2015 . JAK2 activation by growth hormone and other cytokines . Biochem. J . 466 : 1 – 11 . Google Scholar CrossRef Search ADS PubMed Whitmore L. , Wallace B. A. . 2004 . DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data . Nucleic Acids Res. 32 : W668 – W673 . Google Scholar CrossRef Search ADS PubMed Zadworny D. , Shimada K. , Ishida H. , Sato K. . 1989 . Gonadotropin-stimulated estradiol production in small ovarian follicles of the hen is suppressed by physiological concentrations of prolactin in vitro . Gen. Comp. Endocrinol. 74 : 468 – 473 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
Poultry Science – Oxford University Press
Published: Sep 1, 2018
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.
All for just $49/month
Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly
Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.
Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.
Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.
All the latest content is available, no embargo periods.
“Hi guys, I cannot tell you how much I love this resource. Incredible. I really believe you've hit the nail on the head with this site in regards to solving the research-purchase issue.”Daniel C.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud
“I must say, @deepdyve is a fabulous solution to the independent researcher's problem of #access to #information.”@deepthiw
“My last article couldn't be possible without the platform @deepdyve that makes journal papers cheaper.”@JoseServera