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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.


Purification of hmPRα protein by Ni-NTA and amino cellulose column chromatography.(A) Chromatogram and the SDS-PAGE and the western blot analysis results of the Ni-NTA column chromatography fractions obtained from the first purification step. (B) Chromatogram and the SDS-PAGE and western blot analysis results from the Cellufine Amino column chromatography conducted as the second purification step. The elution profile was monitored by absorbance at 280 nm. The horizontal bars in the chromatogram represent the fractions collected for further steps.
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pone.0138739.g003: Purification of hmPRα protein by Ni-NTA and amino cellulose column chromatography.(A) Chromatogram and the SDS-PAGE and the western blot analysis results of the Ni-NTA column chromatography fractions obtained from the first purification step. (B) Chromatogram and the SDS-PAGE and western blot analysis results from the Cellufine Amino column chromatography conducted as the second purification step. The elution profile was monitored by absorbance at 280 nm. The horizontal bars in the chromatogram represent the fractions collected for further steps.

Mentions: In the first step of purification, the sample was separated on a Ni-NTA column. The protein content of the eluted fractions were analyzed by CBBR and immunoblotting with anti-His-tag antibodies. HmPRα protein was detected in fractions 11 to 16 (Fig 3A), which corresponded to 160 mM imidazole in the buffer. These fractions were pooled and applied to a Cellufine Amino column, which we selected as an effective resin for purification of the mPRα protein [24]. The proteins were eluted by linear gradient of sodium chloride (Fig 3B). In the third purification step, the hmPRα fractions were passed through a SP-Sepharose column. The purified hmPRα proteins were concentrated using Cellufine Amino resin. The SDS-PAGE and immunoblotting assay indicated that hmPRα was successfully purified with higher purity (Fig 4A).


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

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

Purification of hmPRα protein by Ni-NTA and amino cellulose column chromatography.(A) Chromatogram and the SDS-PAGE and the western blot analysis results of the Ni-NTA column chromatography fractions obtained from the first purification step. (B) Chromatogram and the SDS-PAGE and western blot analysis results from the Cellufine Amino column chromatography conducted as the second purification step. The elution profile was monitored by absorbance at 280 nm. The horizontal bars in the chromatogram represent the fractions collected for further steps.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138739.g003: Purification of hmPRα protein by Ni-NTA and amino cellulose column chromatography.(A) Chromatogram and the SDS-PAGE and the western blot analysis results of the Ni-NTA column chromatography fractions obtained from the first purification step. (B) Chromatogram and the SDS-PAGE and western blot analysis results from the Cellufine Amino column chromatography conducted as the second purification step. The elution profile was monitored by absorbance at 280 nm. The horizontal bars in the chromatogram represent the fractions collected for further steps.
Mentions: In the first step of purification, the sample was separated on a Ni-NTA column. The protein content of the eluted fractions were analyzed by CBBR and immunoblotting with anti-His-tag antibodies. HmPRα protein was detected in fractions 11 to 16 (Fig 3A), which corresponded to 160 mM imidazole in the buffer. These fractions were pooled and applied to a Cellufine Amino column, which we selected as an effective resin for purification of the mPRα protein [24]. The proteins were eluted by linear gradient of sodium chloride (Fig 3B). In the third purification step, the hmPRα fractions were passed through a SP-Sepharose column. The purified hmPRα proteins were concentrated using Cellufine Amino resin. The SDS-PAGE and immunoblotting assay indicated that hmPRα was successfully purified with higher purity (Fig 4A).

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.