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Expression and Purification of Human Membrane Progestin Receptor α (mPRα).

Hossain MB, Oshima T, Hirose S, Wang J, Tokumoto T - PLoS ONE (2015)

Bottom Line: These results indicated that the hmPRα expressed in yeast was active.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 results indicated that we succeeded in solubilizing and purifying hmPRα in an active form.

View Article: PubMed Central - PubMed

Affiliation: Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8529, Japan.

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.

No MeSH data available.


Characterization of binding activity in membrane fractions.(A) Specific binding activity of [3H]1,2,6,7-progesterone to membrane preparations from untransformed yeast cells (X-33) and hmPRα-producing cells (mPRα-X33). (B) Saturation curves and Scatchard plots of specific [3H]1,2,6,7-progesterone binding to membrane preparations from hmPRα-producing cells (mPRα-X33).
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pone.0138739.g002: Characterization of binding activity in membrane fractions.(A) Specific binding activity of [3H]1,2,6,7-progesterone to membrane preparations from untransformed yeast cells (X-33) and hmPRα-producing cells (mPRα-X33). (B) Saturation curves and Scatchard plots of specific [3H]1,2,6,7-progesterone binding to membrane preparations from hmPRα-producing cells (mPRα-X33).

Mentions: To demonstrate specific binding of [3H]1,2,6,7-progesterone to the expressed hmPRα protein, digitonin was used for the preparation of the cell membrane fraction because this glycoside facilitates steroid receptor access [25,26]. Previously, a final concentration of 0.1% digitonin was reported to be optimal for facilitating steroid binding [24], which was measured using a filter-binding assay [25,26]. After the treatment of the crude cell membrane fractions with 0.1% digitonin, the specific [3H]1,2,6,7-progesterone-binding activity was significantly increased in membrane fractions from hmPRα-expressing cells. Under the same conditions, fractions from untransformed host cells exhibited lower binding activity (Fig 2A). Saturation analysis demonstrated that the progesterone binding to the cell membranes of hmPRα-expressing cells is saturable and of limited capacity (Bmax = 288.8 fmol/mg). Scatchard analysis indicated the presence of a single site of high-affinity binding sites (Kd = 3.8 nM) in the cell membrane fraction of hmPRα (Fig 2B). Consequently, these results indicated that the heterologously produced recombinant hmPRα was active.


Expression and Purification of Human Membrane Progestin Receptor α (mPRα).

Hossain MB, Oshima T, Hirose S, Wang J, Tokumoto T - PLoS ONE (2015)

Characterization of binding activity in membrane fractions.(A) Specific binding activity of [3H]1,2,6,7-progesterone to membrane preparations from untransformed yeast cells (X-33) and hmPRα-producing cells (mPRα-X33). (B) Saturation curves and Scatchard plots of specific [3H]1,2,6,7-progesterone binding to membrane preparations from hmPRα-producing cells (mPRα-X33).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4580469&req=5

pone.0138739.g002: Characterization of binding activity in membrane fractions.(A) Specific binding activity of [3H]1,2,6,7-progesterone to membrane preparations from untransformed yeast cells (X-33) and hmPRα-producing cells (mPRα-X33). (B) Saturation curves and Scatchard plots of specific [3H]1,2,6,7-progesterone binding to membrane preparations from hmPRα-producing cells (mPRα-X33).
Mentions: To demonstrate specific binding of [3H]1,2,6,7-progesterone to the expressed hmPRα protein, digitonin was used for the preparation of the cell membrane fraction because this glycoside facilitates steroid receptor access [25,26]. Previously, a final concentration of 0.1% digitonin was reported to be optimal for facilitating steroid binding [24], which was measured using a filter-binding assay [25,26]. After the treatment of the crude cell membrane fractions with 0.1% digitonin, the specific [3H]1,2,6,7-progesterone-binding activity was significantly increased in membrane fractions from hmPRα-expressing cells. Under the same conditions, fractions from untransformed host cells exhibited lower binding activity (Fig 2A). Saturation analysis demonstrated that the progesterone binding to the cell membranes of hmPRα-expressing cells is saturable and of limited capacity (Bmax = 288.8 fmol/mg). Scatchard analysis indicated the presence of a single site of high-affinity binding sites (Kd = 3.8 nM) in the cell membrane fraction of hmPRα (Fig 2B). Consequently, these results indicated that the heterologously produced recombinant hmPRα was active.

Bottom Line: These results indicated that the hmPRα expressed in yeast was active.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 results indicated that we succeeded in solubilizing and purifying hmPRα in an active form.

View Article: PubMed Central - PubMed

Affiliation: Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8529, Japan.

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.

No MeSH data available.