Purification of the Goldfish Membrane Progestin Receptor Α (Mprα) Ex- Pressed in Yeast Pichia Pastoris
Total Page：16
File Type：pdf, Size：1020Kb
Biomedical Research (Tokyo) 35 (1) 47-59, 2014 Purification of the goldfish membrane progestin receptor α (mPRα) ex- pressed in yeast Pichia pastoris 1 2 1 1, 2 Takayuki OSHIMA , Ryo NAKAYAMA , Shimi Rani ROY , and Toshinobu TOKUMOTO 1 Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka 422-8529, Japan; 2 Biological Science Course, Graduate School of Science, National University Corpo- ration Shizuoka University, Ohya 836, Suruga-ku, Shizuoka 422-8529, Japan (Received 22 November 2013; and accepted 12 December 2013) ABSTRACT Membrane progestin receptors (mPRs) are key mediators of rapid, nongenomic actions of proges- tins on plasma membranes. We established a procedure for the expression and purification of re- combinant goldfish mPRα using the methylotropic yeast Pichia pastoris. In P. pastoris, the recombinant protein, which carried C-terminal histidine and c-Myc tags, was expressed in an ac- tive form as the receptor for maturation-inducing steroids of fish. Expressed proteins were bound reversibly with a high affinity (Kd = 9.4 nM) at a single binding site that could be saturated. After solubilization of mPRα with n-dodecyl-β-D-maltoside (DDM) from yeast membranes, the recom- binant protein was purified using three different columns: first it was affinity-purified over nickel- nitrilotriacetic acid (Ni-NTA), then bound to a cellulose resin with free amino groups and finally to a column with affinity for the c-Myc epitope. The identity of the purified protein was verified by MALDI-TOF/MS analysis and its capacity to bind progestin remained. Expression and purifi- cation of mPRα protein in its functional form will enable the screening of ligands and the deter- mination of its three dimensional structure. Progestins are important steroids regulating final trihydroxy-4-pregnen-3-one (20β-S) was identified maturation at reproductive tissues in male and fe- in Atlantic croaker (Micropogonias undulatus) and male vertebrates. They can be divided into natural spotted seatrout (Cynoscion nebulous) (22). The and synthetic types. Natural type is found in humans structural differences between human progestin, pro- and certain animals (32). Natural progestin in human gesterone, and fish progestins are that fish progestins is progesterone. In the medical fields, a variety of have multiple hydroxyl groups on the side chain at synthetic progestins are used. In teleost fishes, two positions 17, 20 and 21. In fish, MIS is produced by distinct progestins have been identified as naturally the follicular envelope in response to luteinizing hor- occurring maturation inducing steroids (MIS), which mone (LH) from the pituitary (15). In salmon, LH control oocyte maturation. 17α,20β-dihydroxy-4- stimulates a number of signaling cascades leading to pregnen-3-one (17,20β-DHP) was identified in ama- the production of 17α-hydroxyprogesterone (17α-HP) go salmon (Oncorhynchus rhodurus) and 17α,20β,21- from progesterone and 17α-hydroxypregnenolone in the theca cells. Then, 17α-HP is converted to 17,20β-DHP by 20β-hydroxysteroid dehydrogenase Address correspondence to: Toshinobu Tokumoto, PhD, (20β-HSD) in the granulosa cells (18, 22). The Integrated Bioscience Section, Graduate School of Sci- ence and Technology, National University Corporation membrane progestin receptor (mPR) mediates the Shizuoka University, Ohya 836, Suruga-ku, Shizuoka nongenomic actions of progestins on the plasma 422-8529, Japan membrane. It was identified in teleost, a ray-finned Tel & Fax: +81-54-238-4778 fish, at first and subsequently identified in other -ver E-mail: [email protected] tebrates including human (44, 45). Research into the 48 T. Oshima et al. functions of mPR have revealed that this receptor cytes, suggesting that the mPRα and β proteins me- mediates the action of steroids that play a role in, diate the action of maturation inducing steroids. for example, oocyte maturation in fish (13, 21, 35, Membranes prepared from breast cancer cells trans- 37, 42). Genome wide phylogenetic analyses have fected with mPRα exhibited single binding sites that shown that mPRs can be classified into a new pro- were specific for a steroid that induced oocyte matu- tein family, the progestin and adipoQ receptor ration in goldfish, 17,20β-DHP (38). We found that (PAQR) family and they have three subtypes named treatment of oocytes with diethylstilbestrol (DES), mPRα, β and γ (corresponding to PAQR7, 8 and 5, an endocrine disrupting chemical, induced their mat- respectively) (33, 36). The mRNAs of mPRs in hu- uration in both goldfish and zebrafish (39). DES man show differential expression in reproductive tis- also bound with high affinity to membranes from sues such as the ovary, testis, placenta, uterus and mPRα-transfected breast cancer cells. These results nonreproductive tissues such as brain, kidney and suggested that mPR proteins are possibly targeted intestinal tissues, suggesting they mediate different by chemicals that affect the endocrine system. nongenomic actions of progestins (35). In goldfish, Recombinant mPR proteins have been expressed mPRs are also expressed in reproductive tissues, in Escherichia coli and several human cancer cell brain, kidney, eye, gill and spleen. The wide tissue lines (38). Although large-scale culturing in E. coli distribution of mPRs suggests mPRs mediates the is possible, post-translational modification required multiple progestin actions in a wide variety of target to obtain an active form of the recombinant protein tissues (37). Recently, based on the analyses of pro- will not occur. On the other hand, expression of teins expressed in yeast, mPRδ and ε (PAQR6 and 9) mPRs in mammalian cell culture, which would re- were proposed to form novel mPR subtypes (31). sult in proteins that are naturally post-translationally Also human orthologs of them, when expressed in modified, will only reach low levels, which is not human breast cancer cells, were found to have pro- suitable for the purification of the amounts of pro- gestin binding activity (24). Therefore, five mPR tein needed for biochemical and structural analysis. subtypes have been described at present. We therefore selected the methanol-utilizing yeast Rapid nongenomic effects of progestin and mPR Pichia pastoris as a host for the expression of mPRα. expression have been investigated in a wide variety P. pastoris has been used for the expression of G of tissues of fishes and mammals. In addition to protein coupled receptors (GPCRs) and can express mPRs, there are two receptor candidates for mediat- proteins in their functional form in large-scale puri- ing progestin signaling from the cell surface: nucle- fications, as has been described in more than 100 ar progestin receptors (nPRs) and progestin receptor reports. For example, mammalian GPCRs expressed membrane components (PGRMCs). Since their tis- in P. pastoris exhibited a natural ligand binding ac- sue-specific expression overlaps, it seems likely that tivity (19, 43). In this way, P. pastoris has been their functions do as well. Thus, physiological roles used to isolate proteins in amounts that enabled of receptors mediating progestin actions might be structural and biochemical studies of GPCRs (3). In complicated and difficult to unravel (12, 17, 46). 2011 and 2012, the structure for two human GPCRs For example, in addition to its function as a nuclear (the histamine H1 and the adenosine A2a receptor) transcription factor, nPR modulates cell signaling could be determined for the first time after being pathways by activating c-Src and downstream purified from P. pastoris (14, 30). MAPK outside the nucleus (5, 27). In Xenopus oo- In the present study, we established a procedure cytes, like mPRβ, nPR was shown to mediate the for the expression and purification of mPR, a new nongenomic action of progestin during oocyte matu- member of the GPCR family. Recombinant goldfish ration, suggesting a potential interaction between mPRα with progestin binding activity could be ex- mPRs and nPRs (46). Also, transactivation of nPRs pressed in P. pastoris as a fusion-protein with the by the activation of mPRs was demonstrated in hu- pre-pro signal sequence of α-factor from Saccharo- man myometrium (16). Although the details about myces cerevisiae to ensure localization to the plas- the signaling pathway through mPRs still remain ma membrane. The protein was biochemically active unclear, several pathways different from those in- with respect to the binding of a typical mPRα-ligand volving nPRs have been suggested (8, 11, 47). and could be purified by sequential column chroma- We cloned and characterized four mPR subtypes, tography after solubilization from the cell-mem- mPRα, β, γ-1 and γ-2 from goldfish (37, 40). Micro- brane. The described procedure can be used to injection of antisense oligonucleotides targeting generate sufficient amounts of goldfish mPRα pro- mPRα and β blocked the maturation of goldfish oo- tein that would allow the determination of its struc- Purification of recombinant mPRα 49 ture, the analysis of intermolecular interactions, and medium in a 500 mL baffled flask which was incu- the production of monoclonal antibodies. bated at 30°C for 1 day. For induction of mPRα- expression by methanol, cells were harvested by centrifugation, and taken up in 1 L of BMMY medi- MATERIALS AND METHODS um (1% yeast extract, 2% bactopeptone, 100 mM P. pastoris strains expressing goldfish mPRα. For potassium phosphate, pH 6.0, 1.34% yeast nitrogen expression in P. pastoris, the cDNA for goldfish base without amino acids, 4 × 10−5% biotin, 0.5% mPRα, isolated from a mammalian expression vec- methanol) to an OD600 of 1.0–3.0. The medium was tor (38), was fused to the pre-pro secretion signal of placed in a 2 L baffled flask and incubated at 20°C α-factor from S. cerevisiae in the P. pastoris expres- for one day, after which the cells were pelleted at sion vector pPICZαC (Invitrogen). The ORF region 3,000 × g for 5 min, frozen with liquid nitrogen and of the resulting expression cassette (Fig.