Purification of the functional plant membrane channel KAT1

Takao Hibi; Shiho Aoki; Keisuke Oda; Shintaro Munemasa; Shunsuke Ozaki; Osamu Shirai; Yoshiyuki Murata; Nobuyuki Uozumi

doi:10.1016/j.bbrc.2008.07.026

Purification of the functional plant membrane channel KAT1

Takao Hibi, Shiho Aoki, Keisuke Oda, Shintaro Munemasa, Shunsuke Ozaki, Osamu Shirai, Yoshiyuki Murata, Nobuyuki Uozumi

Research output: Contribution to journal › Article › peer-review

5 Citations (Scopus)

Abstract

The inward-rectifying K⁺ channel KAT1 is expressed mainly in Arabidopsis thaliana guard cells. The purification of functional KAT1 has never been reported. We investigated the extraction of the plant K⁺ channel KAT1 with different detergents, as an example for how to select detergents for purifying a eukaryotic membrane protein. A KAT1-GFP fusion protein was used to screen a library of 46 detergents for the effective solubilization of intact KAT1. Then, a "test set" of three detergents was picked for further analysis, based on their biochemical characteristics and availability. The combination use of the selected detergents enabled the effective purification of functional KAT1 with affinity and gel-filtration chromatography.

Original language	English
Pages (from-to)	465-469
Number of pages	5
Journal	Biochemical and Biophysical Research Communications
Volume	374
Issue number	3
DOIs	https://doi.org/10.1016/j.bbrc.2008.07.026
Publication status	Published - Sept 26 2008

Keywords

Affinity chromatography
Detergent screening
Functional purification
Green fluorescent protein
Membrane protein
Potassium channel

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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10.1016/j.bbrc.2008.07.026

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title = "Purification of the functional plant membrane channel KAT1",

abstract = "The inward-rectifying K+ channel KAT1 is expressed mainly in Arabidopsis thaliana guard cells. The purification of functional KAT1 has never been reported. We investigated the extraction of the plant K+ channel KAT1 with different detergents, as an example for how to select detergents for purifying a eukaryotic membrane protein. A KAT1-GFP fusion protein was used to screen a library of 46 detergents for the effective solubilization of intact KAT1. Then, a {"}test set{"} of three detergents was picked for further analysis, based on their biochemical characteristics and availability. The combination use of the selected detergents enabled the effective purification of functional KAT1 with affinity and gel-filtration chromatography.",

keywords = "Affinity chromatography, Detergent screening, Functional purification, Green fluorescent protein, Membrane protein, Potassium channel",

author = "Takao Hibi and Shiho Aoki and Keisuke Oda and Shintaro Munemasa and Shunsuke Ozaki and Osamu Shirai and Yoshiyuki Murata and Nobuyuki Uozumi",

note = "Funding Information: The authors thank Drs. T. Kaidoh and M. Takahashi for their excellent technical assistance in cell culture and antibody production. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.",

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doi = "10.1016/j.bbrc.2008.07.026",

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AU - Hibi, Takao

AU - Aoki, Shiho

AU - Oda, Keisuke

AU - Munemasa, Shintaro

AU - Ozaki, Shunsuke

AU - Shirai, Osamu

AU - Murata, Yoshiyuki

AU - Uozumi, Nobuyuki

N1 - Funding Information: The authors thank Drs. T. Kaidoh and M. Takahashi for their excellent technical assistance in cell culture and antibody production. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

PY - 2008/9/26

Y1 - 2008/9/26

N2 - The inward-rectifying K+ channel KAT1 is expressed mainly in Arabidopsis thaliana guard cells. The purification of functional KAT1 has never been reported. We investigated the extraction of the plant K+ channel KAT1 with different detergents, as an example for how to select detergents for purifying a eukaryotic membrane protein. A KAT1-GFP fusion protein was used to screen a library of 46 detergents for the effective solubilization of intact KAT1. Then, a "test set" of three detergents was picked for further analysis, based on their biochemical characteristics and availability. The combination use of the selected detergents enabled the effective purification of functional KAT1 with affinity and gel-filtration chromatography.

AB - The inward-rectifying K+ channel KAT1 is expressed mainly in Arabidopsis thaliana guard cells. The purification of functional KAT1 has never been reported. We investigated the extraction of the plant K+ channel KAT1 with different detergents, as an example for how to select detergents for purifying a eukaryotic membrane protein. A KAT1-GFP fusion protein was used to screen a library of 46 detergents for the effective solubilization of intact KAT1. Then, a "test set" of three detergents was picked for further analysis, based on their biochemical characteristics and availability. The combination use of the selected detergents enabled the effective purification of functional KAT1 with affinity and gel-filtration chromatography.

KW - Affinity chromatography

KW - Detergent screening

KW - Functional purification

KW - Green fluorescent protein

KW - Membrane protein

KW - Potassium channel

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Purification of the functional plant membrane channel KAT1

Abstract

Keywords

ASJC Scopus subject areas

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