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.
Highlights
Progestins act as a key regulating factor in controlling the reproductive tissues
The physiological effects of progesterone have been known to be mediated by the regulation of gene expression associated with nuclear progesterone receptors [3], new insight on the activity of progesterone was provided by the identification of membrane progestin receptors [4]
Wild yeast P. pastoris was used for the expression and production of recombinant human mPRα protein (hmPRα) protein
Summary
Progestins act as a key regulating factor in controlling the reproductive tissues. Progesterone was identified as a natural progestin in the human body [1]. Synthetic progestins have been produced and are frequently used for medical purposes. Progesterone is a well-known steroid that is produced by the ovary depending on the physiological conditions of the ovary and gonadotropin levels [2]. The physiological effects of progesterone have been known to be mediated by the regulation of gene expression associated with nuclear progesterone receptors [3], new insight on the activity of progesterone was provided by the identification of membrane progestin receptors (mPRs) [4]. Certain nongenomic effects of progestins, such as oocyte maturation, are mediated by mPRs on the plasma
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