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29УДК 636.2:612.6.02:577.175.327:57.083.38
CLONING AND BACTERIAL EXPRESSION OF ALPHA- AND BETA SUBUNITS OF THE FOLLICLE STIMULATING HORMONE
Bursakov S.A., Kovalchuk S.N., Popov D.V., Kosovsky G.Yu.
Center of Experimental Embryology and Reproductive Biotechnologies, Moscow, Russian Federation
ABSTRACT. The article describes the construction of vectors and the carrying out of bacterial expression of recombinant FSH of bovine animals (bFSH) for the production of milligram quantities of its protein subunits. Glycosilated protein bovine follicle stimulating hormone (bFSH) is one of the gonadotropin family, playing important role in reproduction and can be obtained in different systems for heterological expression to preserve its native structure. E. coli was chosen as a host for production of milligram quantities of this protein, because even nonglycosilated bacterial-derived form of the recombinant bFSH to a certain extent preserves biological activity. The major part of alfa and beta subunits of FSH protein is synthesized in form of inclusion bodies, regardless of type of the vector (pET-28a(+) and pET40b(+)) and temperature of expression used (16°C and 37°C). Biosynthesis in the form of inclusion bodies requires that they be solubilized in denatured conditions with following protein refolding into the right shape to function. The washing of inclusion bodies with following ammonium sulphate fractionation leads to 80% pure protein. Simultaneous expression of separate subunits in the same host cell is possible and has some advantages. Non-glycosylated bFSH, as well as its individual subunits, can be used in further studies, including research of their native conformation and biological action, with the aim of developing new effective approaches for improving reproductive technologies in livestock.
Key words: cattle, embryo transplantation, cloning and expression of follicle-stimulating hormone subunits, methods of research
Problemy biologii productivnykh zhivotnykh - Problems of Productive Animal Biology, 2017, 4: 104-110
Introduction
Follicle stimulating hormone (FSH) is a heterodimeric pituitary-derived glycoprotein hormone comprising a a and P subunit produced from different genes. Both a and P polypeptide subunits are synthesized separately and then non-covalently associated into a single entity. The a subunit is identical and conserved within family of gonadotropin glycoprotein hormones, included FSH, luteinizing hormone (LH), chorionic gonadotropin (HCG) and thyroid stimulating hormone (TSH), whereas the unique P subunit is different and confers receptor specificity (Pierce and Parsons 1981; Cahoreau 2015). Both subunits are N-glycosylated and, a-subunit contains five disulfide bonds, whereas each of the p-subunits in this family of hormones contains six disulfide bonds that are located in the same relative positions (Bursakov et al., 2016). Formation of specific disulfide bonds is essential for stabilization of bFSH folding intermediates and for achieving its assembly competent state (Sone et al., 1997). Intracellular levels of free a subunits are greater than those of the mature glycoprotein, implying that the synthesis of a and p subunits is independently regulated and the hormone assembly is limited by the appearance of the specific p subunit (Goodwin et al., 1983).
FSH is responsible for growth and maturation of follicles in females and steroidogenesis in males. bFSH is used in the cattle industry as an integral part of superovulation protocol to trigger the development of more than one dominant follicle. Recombinant bFSH could provide a safe alternative, which are devoid contaminating pituitary hormones for superovulation protocol. A suitable recombinant form of FSH is not readily available in the cattle industry, but many advanced research
has been made already in this direction to completely modify current superovulation protocol and eliminate any opportunities for contamination and disease transmission. A variety of methods for production of recombinant FSH for animal husbandry has increased significantly over the last 30 years, but so far failed to develop a common standard, that is consistent with the existing requirements to be included in protocol for induction of superovulation in cattle on a permanent basis and for obtaining the expected number of embryos per donor animal (Bursakov et al., 2016).
Recombinant bovine bFSH was produced from many prokaryotic and eukaryotic sources. E.coli is one of the earliest and the most widely used hosts for the production of heterologous recombinant proteins and has been successfully used for both industrial- and laboratory- scale cytosolic production of recombinant proteins. However, there are often problems in recovering substantial yields of correctly folded proteins in E. coli (Strickland et al., 1985; Wilson et al., 2008; Rogan et al., 2009; Zhao et al., 2010; Hesser 2011; Vashitha et al., 2013). Besides, it is widely accepted that post-translational glycosilations to the FSH molecule are important for in vivo half-life and must be present on the recombinant form to be most effective as a treatment. The lack of proper glycosilations is what has limited use of non-eukaryotic heterologous protein expression systems, as there are concerns that the protein will not be active long enough in vivo to cause desired effects.
The importance of carbohydrate in the folding pathway of gonadotropins is still incompletely understood. The lack of carbohydrate could affect the ability of bFSH to achieve or maintain a biologically active conformation that could hinder the production of biologically active bFSH in bacterial cells. However, the lack of glycosilation of the recombinant protein that may decrease efficiency of folding, did not eliminate biological activity, as the ovarian weights in treated cells increased after delivery of bacterial-derived recombinant bFSHpa (Hesser 2011). Though, a nonglycosilated bacterial-derived form of recombinant bFSH may be used to stimulate ovarian development.
The aim of this study was to construct the vectors for the expression of recombinant bFSH for production of milligram quantities of its protein subunits.
Materials and methods
Isolation of total RNA and synthesis of cDNA. Two pituitary glands of caw were obtained from the local slaughter house. The tissues were cutted in small peaces and frozen in liquid nitrogen immediately after dissection, and then stored at -80°C until analysis.
Isolation of total RNA and cDNA synthesis was made according to the instruction of Evrogen (BC032). Total RNA was extracted from 1/8 pituitary gland using Extract RNA reagent, following to the manufacturer instructions. Final total RNA was dissolved in 0.1% DEPC-treated water and frozen -20°C until use. RNA quality was checked by electrophoresis using an ethidium bromide-stained agarose gel, and RNA concentrations were determined with a BioPhotometer plus (Eppendorf AG, Germany) at 260 nm. Quality of total RNA was tested on the agarose gel by presence of equal ratio 1:1 between 28S (4500 bp) h 18S (1900 bp) RNA. RNA samples with A260nm/A280nm ratios of 1.8 to 2.0 were used for further analysis.
First strand cDNA was synthesized according to MMLV RT kit (SK021) protocol of Evrogen from 1 ^g of total RNA, using a special oligo(dT) primer and MMLV revertase. cDNA was used as a template for PCR amplification with a gene specific primers 1PF (5'-GAAGAATTCtttcctgatggagagtttac-3'), 2PR 5'-GTTGCGGCCGCTTAggatttgtgataataacaagtgctgca -3' for a-subunit, and 3PF (5'-GTCGAATTCtgcgagctgaccaacatcac-3'), 4PR 5'-GTTGCGGCCGCttattctttgatttccctgaaggag-3') for p-subunit and restriction sites for EcoRI and NotI for cloning in pET-28a(+). FKpnIb1 (5'-
TGGTACCGATGACGACGACAAGCGCAGCTGCGAGCTGACCAACATCAC-3') and
RNcoIendb2 (5'-GGGTTCCATGGTTATTctttgatttccctgaaggagc-3') for beta subunit and FKpnIa1 (5 '-TGGTACCGATGACGACGACAAGTTTCCTGATGGAGAGTTTACAATGC-3') and
RNcoIenda2 (5 '-GGGTTCCATGGTTAGGATTTGTGATAATAACAAGTGCT-3') for alfa subunit to obtain full-length FSHa and FSHp and for cloning in pET-40b(+). The 5'-sense primers for cloning in pET-40b(+) were designed without the coding region for mammalian signal sequence. All
primers were designed equally with the sites for Kpnl and Ncol and stop codon to ensure translation termination.
Thermal cycling was performed on a thermalcycler (NixTechnick, Ink) using the following programme for FSHa and FSHp - 98°C for 2 min, and 35 cycles, where each cycle consisted of 98°C for 20 s, 53°C-62°C (depended of pair of primers) for 20 s, 72°C for 30 s; and 72°C for 10 min. The PCR products were cloned into pET-28a(+) and pET-40b(+) vectors, and recombinant plasmids were transformed into E. coli competent cells (strain DH5a). PCR products were separated by agarose gel electrophorese and purified using a gel purification kit (Cleanup Mini, BC023, Evrogen, Russia). DNA was diluted and digested with 10U of restriction endonucleases for 2 h. The digested DNA insert was purified on 1% low melting agarose gel, stained with ethidium bromide, and excised from the gel for use in ligation reaction. To prepare the pET-28a(+) and pET-40b(+) vectors for ligation, the 3 ^g of each plasmid were digested with required pair of corresponding endonucleases 10U in appopriate buffer for 2 h at 37°C. The linearized plasmid was purified on a 1% low melting agarose gel, stained with ethidium bromide, and excised from the gel. The liniarized vector and digested FSHa or FSHp genes were mixed with 0.5 U T4 DNA ligase in 10 ^l reaction mixture.
The ligation reaction was performed overnight at 16°C and was then used to transfer E. coli DH5a cells made competent by the CaCl2 method.
Cloning and bacterial expression alpha and beta subunits of FSH. Colonies were isolated from Luria-Bertani agar plates containing 170 ^g/ml chloramphenicol and 50 ^g/ml kanamicin and used to prepare 3 ml cultures in Luria-Bertani broth. Plasmids were purified from these cultures and screened for the presence of the FSH gene by PCR analysis. Plasmid from a positive clone was used to transform E. coli BL21 (DE3) competent cells.
Transformed E. coli BL21(DE3) colonies that contained the bFSHa/p plasmid were grown up overnight in LB medium under the control of two antibiotics - kanamicin (50 ^g/ml) and chloramphenicol (170 ^g/ml). Cells suspensions were diluted 1:100 in 200 ml of LB medium containing antibiotics and grown at 37°C. Expression was initiated by addition of 0.4 mM IPTG in the mid-log phase of cell growth (0.4-0.7 absorbance at 600 nm). Cultivation was continued for 6 hrs at the same temperature. Biomass was collected by centrifugation at 3000 g and resuspended in 15 ml of 20 mM TrisHCl buffer, pH 8 with 2 M NaCl and 10 mM dithiothreitol (DTT).
Cells were disrupted by sonication twice by 10 min at 22 kHz with presence of 1 mM serine protease inhibitor - phenylmethylsulfonyl fluoride (PMSF). After centrifugation (5900g) pellet was solubilized in 50 mM TrisHC buffer, pH8 with 1 mM DTT in presence of 6 M urea.
The solubilized subunits of FSH were reconstructed during prolonged dialysis against 50 mM TrisHC buffer, pH 8 with 3 times buffer exchange - (1). 1.5 M urea, 1 mM DTT and oxidized glutathione (GSH, 0,1 mM, 15 h; (2). 50 mM TrisHC buffer, pH 8, 6 h; (3). 50 mM TrisHC buffer, pH 8, 14 h. Finally urea concentration was diminished completely during protein concentration on the centricon Mw 10000 (Amicon, Millipore, USA).
Results and discussion
To create the plasmid constructs for obtaining recombinant bFSH, the coding portions DNA of the a and p subunits were amplified using primers comprising a restriction site elongated with three nucleotides for the reliable functioning of the restriction enzymes. In addition, two pairs of primers composed of the mature coding portion of the bFSH of both subunits, and the enterokinase site preceding the 6-membered histidine peptide (His-tag) were also created for the further purification of the resulting protein using metal chelating chromatography on a Ni-column.
For expression of recombinant bFSH in E. coli, two different vectors pET-28b(+) and pET-40b(+) were used. Last one designed for expression of DsbC fusion protein. In case of both expression systems, most of bFSHa and bFSHp protein was expressed in the form of inclusion bodies. The pET Dsb Fusion Systems 40b are designed for cloning and high-level expression of peptide sequences fused with 236-aa DsbC^Tag™ sequence [pET-40b(+)]. DsbC are periplasmic enzyme that cata-
lyzes isomerization of disulfide bonds (Rietsch, 1996; Sone 1997; Missiakas, 1994; Zapun, 1995; Raina 1997). DsbC^Tag vector enable potential periplasmic localization and thus may enhance solubility and proper folding in this non-reducing environment (Collins-Racie, 1995). The prokaryotic periplasm provides oxidizing conditions for disulfide bonding; however, inclusion body formation can also occur in the periplasm (Georgiou et al., 1986).
Using restriction enzymes EcoRI and NotI for pET-28a (+) and KpnI and NcoI for vector pET-40b(+), the coding portions of the subunits were inserted separately into the bacterial vector for protein expression. Thus, we developed and obtained plasmid vectors for the heterologous expression of alpha and beta subunits of FSH in E. coli BL21 (DE3) cells.
The electrophoresis data of E. coli whole cells before and after IPTG induction are shown in Fig. 1, 2. The expression level of recombinant bFSH is higher than 20% of the total E. coli protein. Small scale expression cultures revealed that adequate amounts of recombinant bFSHa, bFSHp, and bFSHa+bFSHp protein were produced after 6 hrs of induction in presence of 0.4 mM IPTG (Fig. 1, 2). However, only in case of pET-40b(+) vector small quantity of protein bFSHa and bFSHp appeared in soluble fraction. In most cases, inclusion body formation is a consequence of high expression rates, regardless of the system or protein used (Lilie et al., 1998).
The relative yield of soluble protein increased with a decreased rate of protein synthesis. Whereas recombinant proteins often aggregate when E. coli cells are cultivated at 37°C, reduction of the cultivation temperature can increase the amount of native protein due to a decrease of the rate of protein synthesis (Schein, Noteborn, 1988). The change of the temperature from 37°C to 16°C for cultivation does not change much the yield of the searching protein in case of bFSH.
Figure 1. bFSH induction by different IPTG concentrations (line 1 - 0; line 2 - 0.1; line 3 -0.4; line 4 - Standard (markers masses shown on the left side of the gel); line 5 - 1 mM IPTG, bFSHa; line 6 - 0; line 7 - 0.1; line 8 - 0.4; line 9 - 1 mM IPTG bFSH ft). Expressed protein position marked by triangle.
In the case of a simple and efficient renaturation procedure, deposition of the protein in inclusion bodies and subsequent isolation and renaturation of inclusion body protein often means the most straightforward strategy to get large amounts of recombinant protein. The primary purification step is centrifugation of disrupted cells that removes soluble E. coli proteins. The further treatment of
the precipitate with 2 M NaCl provides further purification of the inclusion bodies from soluble proteins.
Figure 2. bFSHa+p induction by different IPTG concentrations (line 1 - 0; line 2 - 0.1; line 3 - 0.4; line 4 -1 mMIPTG; line 5 - Standard (markers masses shown on the right side of the gel). Expressed protein position marked by triangle.
Figure 3. bFSH after 20% ammonium sulphate precipitation, Line 1 - Standard; line 2 - bFSH a; line 3 - bFSHß; line 4 - b FSHa+ß. Expressed protein position marked by triangle.
Correct refolding plays important role in protein structure formation. The renaturation buffer has to be supplemented with a redox system because FSH a and p subunit contains 5 and 6 disulfide bands correspondingly. The addition of a mixture of the reduced and oxidized forms of low molecular weight thiol reagents, such as glutathione, cysteine and cysteamine (molar ratios of reduced to oxidized compounds 1:1 to 5:1, respectively), usually provides the appropriate redox potential to allow formation and reshuffling of disulfides (Burgess, 2009; Basu, et al., 2011).
Accordingly, refolding optimization was attempted because of its high importance. Correspondingly the pair of dithiotreyitol and oxidized glutation (in optimized ratio DTT: GSH(ox) = 10 -1 mM to 0,1 mM) were included during refolding.
The purity of bFSH protein solubilized from inclusion bodies in 6 M urea is sufficient for refolding procedure. The optimal conditions for bFSH refolding were at pH 8 and 1.8 M urea and with changed ratio for DTT and oxidized glutathione from 1:7 to 1:5 (0.1 and 0.5 mM respectively). Isolation of the recombinant bFSH from the refolding medium and is subsequent purification by ammonim sulphate precipitation and metal chelating chromatography yields enriched fraction of the protein.
Purification bFSH subunits are performed by precipitation with ammonium sulphate, which concentrates the hormone and provides the removal of the rests of refolding medium components and improperly folded or aggregated protein. Protein preparations obtained after this step were concentrated on the centricon at ~10^g/ml and purity was 80-85% (Fig. 3). For preparative purpose, further purification can be achieved by metal chelating chromatography on Ni-Sepharose column.
Thus, the goal of the current study was to get producers for obtaining recombinant subunits a and p of the bFSH and also both subunits in one host. Structural and functional analyses of proteins,
especially for biological or industrial applications, require large amounts of proteins. Inclusion body production and protein renaturation provides one of the possible efficient routes to meet these requirements and isolate recombinant bFSH. We found that all three variants (a, p, and a+p) in which we are interested are in the similar order of expression quantity, except that a+P variant has its own advantage. It needs a half of effort to get the heterodimeric complex of recombinant bFSH.
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Клонирование и экспрессия альфа- и бета-субъединиц фолликулостимулирующего гормона
Бурсаков С.А., Ковальчук С.Н., Попов Д.В., Косовский Г.Ю.
Центр экспериментальной эмбриологии и репродуктивных биотехнологий, 127422, Москва, Российская Федерация
В статье описано построение векторов и проведение бактериальной экспрессии реком-бинантного ФСГ крупного рогатого скота (бФСГ) для получения миллиграммовых количеств его белковых субъединиц. Один из членов семейства гонадотропинов — гликозилированный белковый бФСГ играет важную роль в размножении и может быть получен в разных системах для гетерологической экспрессии с сохранением своей нативной структуры. Клетки E. coli были выбраны в качестве организма хозяина для производства миллиграммовых количеств этого белка, поскольку даже негликозилированная бактериальная форма рекомбинантного бФСГ в определённой степени сохраняет биологическую активность. Основная часть альфа- и бета-субъединиц белка бФСГ синтезируется в форме телец включения, независимо от типа используемого вектора (pET-28a (+) и pET40b (+)) и используемой температуры экспрессии (16°C и 37°С). Биосинтез в виде телец включения обязывает проводить их солюбилизацию в денатурирующих условиях с последующим рефолдингом белка в правильную для функционирования форму. Промывка телец включения с последующим фракционированием сульфатом аммония приводит к белку с высокой степенью очистки - около 80%. Одновременная экспрессия отдельных субъединиц в одной и той же клетке хозяина возможна и имеет некоторые преимущества. Негликозилированный бФСГ, а также его отдельные субъединицы могут быть использованы в дальнейших исследованиях, в том числе для изучения их нативной кон-формации и биологического действия с целью разработки новых эффективных подходов для совершенствования репродуктивных технологий в животноводстве.
Ключевые слова: крупный рогатый скот, трансплантация эмбрионов, клонирование и экспрессия субъединиц фолликулостимулирующего гормона, методы исследоваия
Проблемы биологии продуктивных животных, 2017, 4: 104-110
Поступило в редакцию: 20.08.2017 Получено после доработки: 11.09.2017
Бурсаков Сергей Алексеевич, к.б.н., с.н.с., тел.: 8(495)610-21-31; [email protected]; Ковальчук Светлана Николаевна, к.б.н., зав. отд., тел.: 8(495)610-21-31; [email protected];
Попов Дмитрий Владимирович, зав. отд., тел.: 8(495)610-21-31; [email protected]; Косовский Глеб Юрьевич, д.б.н., директор, тел.: 8(495)610-21-31, [email protected]
