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.

Proteomic Profile ofMabuyasp. (Squamata: Scincidae) Ovary and Placenta During Gestation

“…Fish membrane progestin receptors homologs have been classified as mPR α, β, and γ and its expression have been identified in different organisms: human, mouse, pig, Xenopus , and zebrafish (Zhu et al., ). The mPRα is widely expressed in different vertebrate species and reproductive tissues (ovary, testis, uterus, and placenta), suggesting its multiple physiological functions (Hossain et al., ). For example, it mediates nongenomic actions of progestins including the secretion of gonadotropin‐releasing hormone, maturation of oocytes in fish, immune modulation, vasodilation, transportation, and relaxation of the uterine myometrium during mammalian pregnancy (Thomas, ; Dressing et al., ).…”

Histone Deacetylase 1 Plays an Acetylation-Independent Role in Influenza A Virus Replication

“…Glutathione S-transferase (GST) fusion protein GST-HDAC1 and 6His fusion protein 6His-NP were expressed in E. coli strain BL21 host cells induced by 0.5 mM IPTG for 4 h at 28°C. After cell lysis and centrifugation, the GST-HDAC1 and 6His-NP proteins were purified with glutathione-sepharose 4B and nickel-nitrilotriacetic acid (Ni-NTA) column (QIAGEN, Gaithersburg, MD, USA) as reported previously ( 31 , 32 ), respectively. The purified proteins were concentrated and dialyzed against PBS.…”

In-vitro prostate cancer biomarker detection by directed conjugation of anti-PSCA antibody to super paramagnetic iron oxide nanoparticless

“…Transformation and expression in competent cells of E. coli BL21 were done as previously described (16). Finally, purification of the His-tagged PSCA protein was performed by a Ni-NTA column (Invitrogen, CA, USA) based on the manufacturer's instructions and tested for purity and immunoreactivity using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie blue (data not shown) and immunoblotting, respectively (17).…”