title: Expression and Purification of Human Membrane Progestin Receptor α (mPRα).
abstract: Membrane progestin receptors (mPRs) are responsible for mediating the rapid, nongenomic activity of progestins and belong to the G protein-coupled receptor (GPCR) family. mPRs are also considered as attractive proteins to draw a new medicinal approach. In this study, we optimized a procedure for the expression and purification of recombinant human mPRα protein (hmPRα) by a methylotropic yeast, Pichia pastoris, expression system. The protein expressed in crude membrane fractions exhibited a binding affinity of Kd = 3.8 nM and Bmax = 288.8 fmol/mg for progesterone. These results indicated that the hmPRα expressed in yeast was active. Solubilized hmPRα was purified through three column chromatography steps. A nickel-nitrilotriacetic acid (Ni-NTA) column was first used, and the mPRα proteins were then bound to cellulose resin with free amino groups (Cellufine Amino) and finally passed through an SP-Sepharose column. The optimization of expression and purification conditions resulted in a high yield of purified hmPRα (1.3-1.5 mg from 1 L culture). The purified hmPRα protein demonstrated progesterone binding (Kd = 5.2 nM and Bmax = 111.6 fmol/mg). The results indicated that we succeeded in solubilizing and purifying hmPRα in an active form. Sufficient amount of active hmPRα protein will support the establishment of applications for the screening of ligands for mPRα.
authors.affiliation: Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8529, Japan.
authors.forename: Md Babul
authors.initials: MB
authors.lastname: Hossain
authors.affiliation: Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8529, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Oshima
authors.affiliation: Department of Biological Science, Faculty of Science, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8529, Japan.
authors.forename: Shizuka
authors.initials: S
authors.lastname: Hirose
authors.affiliation: Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8529, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.affiliation: Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8529, Japan; Department of Biological Science, Faculty of Science, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8529, Japan.
authors.forename: Toshinobu
authors.initials: T
authors.lastname: Tokumoto
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D010843:Pichia / Q000378:metabolism; D011374:Progesterone / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D011980:Receptors, Progesterone; D011994:Recombinant Proteins; C513556:PAQR7 protein, human; D011374:Progesterone