Overexpression and purification of the vanilloid receptor in yeast (Saccharomyces cerevisiae)

Vera Yu Moiseenkova; Helen L. Hellmich; Burgess N. Christensen

doi:10.1016/j.bbrc.2003.09.011

Overexpression and purification of the vanilloid receptor in yeast (Saccharomyces cerevisiae)

Vera Yu Moiseenkova, Helen L. Hellmich, Burgess N. Christensen

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

11 Scopus citations

Abstract

The vanilloid receptor type 1 (VR1) is a novel membrane receptor activated by heat or chemical ligands conveying information about chemosensitive and thermosensitive pain. We have overexpressed and purified wild type VR1 (wtVR1) as well as several mutant forms using the yeast strain Saccharomyces cerevisiae with the goal of obtaining sufficient protein for structural studies. To facilitate the rapid assaying of protein production and purification we used PCR to construct mutant VR1-green fluorescent protein fusion genes. All recombinant inserts were engineered with 12 HIS tags on the C-terminus for metal affinity column purification. The yield of purified protein from 16L fermentation was about 1mg following a single-step purification procedure. By taking advantage of the calcium permeability of VR1 we measured changes in [Ca²⁺]_i in capsaicin-stimulated fura-2 loaded yeast cells expressing VR1.

Original language	English (US)
Pages (from-to)	196-201
Number of pages	6
Journal	Biochemical and Biophysical Research Communications
Volume	310
Issue number	1
DOIs	https://doi.org/10.1016/j.bbrc.2003.09.011
State	Published - Oct 10 2003
Externally published	Yes

Keywords

Fura2
Fusion proteins
Overexpression
Protein purification
VR1
Vanilloid receptor

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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10.1016/j.bbrc.2003.09.011

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title = "Overexpression and purification of the vanilloid receptor in yeast (Saccharomyces cerevisiae)",

abstract = "The vanilloid receptor type 1 (VR1) is a novel membrane receptor activated by heat or chemical ligands conveying information about chemosensitive and thermosensitive pain. We have overexpressed and purified wild type VR1 (wtVR1) as well as several mutant forms using the yeast strain Saccharomyces cerevisiae with the goal of obtaining sufficient protein for structural studies. To facilitate the rapid assaying of protein production and purification we used PCR to construct mutant VR1-green fluorescent protein fusion genes. All recombinant inserts were engineered with 12 HIS tags on the C-terminus for metal affinity column purification. The yield of purified protein from 16L fermentation was about 1mg following a single-step purification procedure. By taking advantage of the calcium permeability of VR1 we measured changes in [Ca2+]i in capsaicin-stimulated fura-2 loaded yeast cells expressing VR1.",

keywords = "Fura2, Fusion proteins, Overexpression, Protein purification, VR1, Vanilloid receptor",

author = "Moiseenkova, {Vera Yu} and Hellmich, {Helen L.} and Christensen, {Burgess N.}",

note = "Funding Information: This work was supported by a Sealy Development grant to BNC. MV was supported in part by the Center for Bioengineering, University of Texas Medical Branch.",

year = "2003",

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doi = "10.1016/j.bbrc.2003.09.011",

language = "English (US)",

volume = "310",

pages = "196--201",

journal = "Biochemical and Biophysical Research Communications",

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publisher = "Academic Press Inc.",

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T1 - Overexpression and purification of the vanilloid receptor in yeast (Saccharomyces cerevisiae)

AU - Moiseenkova, Vera Yu

AU - Hellmich, Helen L.

AU - Christensen, Burgess N.

N1 - Funding Information: This work was supported by a Sealy Development grant to BNC. MV was supported in part by the Center for Bioengineering, University of Texas Medical Branch.

PY - 2003/10/10

Y1 - 2003/10/10

N2 - The vanilloid receptor type 1 (VR1) is a novel membrane receptor activated by heat or chemical ligands conveying information about chemosensitive and thermosensitive pain. We have overexpressed and purified wild type VR1 (wtVR1) as well as several mutant forms using the yeast strain Saccharomyces cerevisiae with the goal of obtaining sufficient protein for structural studies. To facilitate the rapid assaying of protein production and purification we used PCR to construct mutant VR1-green fluorescent protein fusion genes. All recombinant inserts were engineered with 12 HIS tags on the C-terminus for metal affinity column purification. The yield of purified protein from 16L fermentation was about 1mg following a single-step purification procedure. By taking advantage of the calcium permeability of VR1 we measured changes in [Ca2+]i in capsaicin-stimulated fura-2 loaded yeast cells expressing VR1.

AB - The vanilloid receptor type 1 (VR1) is a novel membrane receptor activated by heat or chemical ligands conveying information about chemosensitive and thermosensitive pain. We have overexpressed and purified wild type VR1 (wtVR1) as well as several mutant forms using the yeast strain Saccharomyces cerevisiae with the goal of obtaining sufficient protein for structural studies. To facilitate the rapid assaying of protein production and purification we used PCR to construct mutant VR1-green fluorescent protein fusion genes. All recombinant inserts were engineered with 12 HIS tags on the C-terminus for metal affinity column purification. The yield of purified protein from 16L fermentation was about 1mg following a single-step purification procedure. By taking advantage of the calcium permeability of VR1 we measured changes in [Ca2+]i in capsaicin-stimulated fura-2 loaded yeast cells expressing VR1.

KW - Fura2

KW - Fusion proteins

KW - Overexpression

KW - Protein purification

KW - VR1

KW - Vanilloid receptor

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DO - 10.1016/j.bbrc.2003.09.011

M3 - Article

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SN - 0006-291X

VL - 310

SP - 196

EP - 201

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

IS - 1

ER -

Overexpression and purification of the vanilloid receptor in yeast (Saccharomyces cerevisiae)

Abstract

Keywords

ASJC Scopus subject areas

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