title: Purification of the goldfish membrane progestin receptor α (mPRα) expressed in yeast Pichia pastoris.
abstract: Membrane progestin receptors (mPRs) are key mediators of rapid, nongenomic actions of progestins on plasma membranes. We established a procedure for the expression and purification of recombinant 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 active 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 recombinant protein was purified using three different columns: first it was affinity-purified over nickel-nitrilotriaceticacid (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 purification of mPRα protein in its functional form will enable the screening of ligands and the determination of its three dimensional structure.
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.forename: Ryo
authors.initials: R
authors.lastname: Nakayama
authors.forename: Shimi Rani
authors.initials: SR
authors.lastname: Roy
authors.forename: Toshinobu
authors.initials: T
authors.lastname: Tokumoto
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D006054:Goldfish / Q000235:genetics*; D010843:Pichia / Q000235:genetics*; D011372:Progestins / Q000378:metabolism; D011485:Protein Binding; D011980:Receptors, Progesterone / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
chemical_list: D011372:Progestins; D011980:Receptors, Progesterone; D011993:Recombinant Fusion Proteins