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

Vera Yu Moiseenkova, Helen Hellmich, Burgess N. Christensen

Research output: Contribution to journalArticle

10 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)196-201
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume310
Issue number1
DOIs
StatePublished - Oct 10 2003

Fingerprint

TRPV Cation Channels
Yeast
Purification
Saccharomyces cerevisiae
Yeasts
Proteins
Fura-2
Gene Fusion
Capsaicin
Distillation columns
Conveying
Green Fluorescent Proteins
Fermentation
Permeability
Fusion reactions
Hot Temperature
Genes
Metals
Cells
vanilloid receptor subtype 1

Keywords

  • Fura2
  • Fusion proteins
  • Overexpression
  • Protein purification
  • Vanilloid receptor
  • VR1

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Overexpression and purification of the vanilloid receptor in yeast (Saccharomyces cerevisiae). / Moiseenkova, Vera Yu; Hellmich, Helen; Christensen, Burgess N.

In: Biochemical and Biophysical Research Communications, Vol. 310, No. 1, 10.10.2003, p. 196-201.

Research output: Contribution to journalArticle

@article{1ef5e66e59eb45ad9326b1186e56029d,
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, Vanilloid receptor, VR1",
author = "Moiseenkova, {Vera Yu} and Helen Hellmich and Christensen, {Burgess N.}",
year = "2003",
month = "10",
day = "10",
doi = "10.1016/j.bbrc.2003.09.011",
language = "English (US)",
volume = "310",
pages = "196--201",
journal = "Biochemical and Biophysical Research Communications",
issn = "0006-291X",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

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

AU - Moiseenkova, Vera Yu

AU - Hellmich, Helen

AU - Christensen, Burgess N.

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 - Vanilloid receptor

KW - VR1

UR - http://www.scopus.com/inward/record.url?scp=0141849083&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0141849083&partnerID=8YFLogxK

U2 - 10.1016/j.bbrc.2003.09.011

DO - 10.1016/j.bbrc.2003.09.011

M3 - Article

VL - 310

SP - 196

EP - 201

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 1

ER -