Abstract
Membrane progestin receptors (mPRs) are key mediators of rapid, nongenomic actions of progestinson plasma membranes. We established a procedure for the expression and purification of recombinantgoldfish mPRα using the methylotropic yeast Pichia pastoris. In P. pastoris, therecombinant protein, which carried C-terminal histidine and c-Myc tags, was expressed in an activeform as the receptor for maturation-inducing steroids of fish. Expressed proteins were boundreversibly with a high affinity (Kd = 9.4 nM) at a single binding site that could be saturated. Aftersolubilization of mPRα with n-dodecyl-β-D-maltoside (DDM) from yeast membranes, the recombinantprotein was purified using three different columns: first it was affinity-purified over nickelnitrilotriaceticacid (Ni-NTA), then bound to a cellulose resin with free amino groups and finallyto a column with affinity for the c-Myc epitope. The identity of the purified protein was verifiedby MALDI-TOF/MS analysis and its capacity to bind progestin remained. Expression and purificationof mPRα protein in its functional form will enable the screening of ligands and the determinationof its three dimensional structure.
