Saccharomyces cerevisiae-based platform for rapid production and evaluation of Eukaryotic Nutrient transporters and transceptors for biochemical studies and crystallography

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Saccharomyces cerevisiae-based platform for rapid production and evaluation of Eukaryotic Nutrient transporters and transceptors for biochemical studies and crystallography. / Scharff-Poulsen, Peter; Pedersen, Per Amstrup.

In: PLoS ONE, Vol. 8, No. 10, e76851, 2013.

Research output: Contribution to journalJournal articlepeer-review

Harvard

Scharff-Poulsen, P & Pedersen, PA 2013, 'Saccharomyces cerevisiae-based platform for rapid production and evaluation of Eukaryotic Nutrient transporters and transceptors for biochemical studies and crystallography', PLoS ONE, vol. 8, no. 10, e76851. https://doi.org/10.1371/journal.pone.0076851

APA

Scharff-Poulsen, P., & Pedersen, P. A. (2013). Saccharomyces cerevisiae-based platform for rapid production and evaluation of Eukaryotic Nutrient transporters and transceptors for biochemical studies and crystallography. PLoS ONE, 8(10), [e76851]. https://doi.org/10.1371/journal.pone.0076851

Vancouver

Scharff-Poulsen P, Pedersen PA. Saccharomyces cerevisiae-based platform for rapid production and evaluation of Eukaryotic Nutrient transporters and transceptors for biochemical studies and crystallography. PLoS ONE. 2013;8(10). e76851. https://doi.org/10.1371/journal.pone.0076851

Author

Scharff-Poulsen, Peter ; Pedersen, Per Amstrup. / Saccharomyces cerevisiae-based platform for rapid production and evaluation of Eukaryotic Nutrient transporters and transceptors for biochemical studies and crystallography. In: PLoS ONE. 2013 ; Vol. 8, No. 10.

Bibtex

@article{0c19fb8b257f44bf95fc4ecddafc549e,
title = "Saccharomyces cerevisiae-based platform for rapid production and evaluation of Eukaryotic Nutrient transporters and transceptors for biochemical studies and crystallography",
abstract = "To produce large quantities of high quality eukaryotic membrane proteins in Saccharomyces cerevisiae, we modified a high-copy vector to express membrane proteins C-terminally-fused to a Tobacco Etch Virus (TEV) protease detachable Green Fluorescent Protein (GFP)-8His tag, which facilitates localization, quantification, quality control, and purification. Using this expression system we examined the production of a human glucose transceptor and 11 nutrient transporters and transceptors from S. cerevisiae that have not previously been overexpressed in S. cerevisiae and purified. Whole-cell GFP-fluorescence showed that induction of GFP-fusion synthesis from a galactose-inducible promoter at 15{\^A}°C resulted in stable accumulation of the fusions in the plasma membrane and in intracellular membranes. Expression levels of the 12 fusions estimated by GFP-fluorescence were in the range of 0.4 mg to 1.7 mg transporter pr. liter cell culture. A detergent screen showed that n-dodecyl-{\~A}Ÿ-D-maltopyranoside (DDM) is acceptable for solubilization of the membrane-integrated fusions. Extracts of solubilized membranes were prepared with this detergent and used for purifications by Ni-NTA affinity chromatography, which yielded partially purified full-length fusions. Most of the fusions were readily cleaved at a TEV protease site between the membrane protein and the GFP-8His tag. Using the yeast oligopeptide transporter Ptr2 as an example, we further demonstrate that almost pure transporters, free of the GFP-8His tag, can be achieved by TEV protease cleavage followed by reverse immobilized metal-affinity chromatography. The quality of the GFP-fusions was analysed by fluorescence size-exclusion chromatography. Membranes solubilized in DDM resulted in preparations containing aggregated fusions. However, 9 of the fusions solubilized in DDM in presence of cholesteryl hemisuccinate and specific substrates, yielded monodisperse preparations with only minor amounts of aggregated membrane proteins. In conclusion, we developed a new effective S. cerevisiae expression system that may be used for production of high-quality eukaryotic membrane proteins for functional and structural analysis.",
author = "Peter Scharff-Poulsen and Pedersen, {Per Amstrup}",
year = "2013",
doi = "10.1371/journal.pone.0076851",
language = "English",
volume = "8",
journal = "PLoS ONE",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "10",

}

RIS

TY - JOUR

T1 - Saccharomyces cerevisiae-based platform for rapid production and evaluation of Eukaryotic Nutrient transporters and transceptors for biochemical studies and crystallography

AU - Scharff-Poulsen, Peter

AU - Pedersen, Per Amstrup

PY - 2013

Y1 - 2013

N2 - To produce large quantities of high quality eukaryotic membrane proteins in Saccharomyces cerevisiae, we modified a high-copy vector to express membrane proteins C-terminally-fused to a Tobacco Etch Virus (TEV) protease detachable Green Fluorescent Protein (GFP)-8His tag, which facilitates localization, quantification, quality control, and purification. Using this expression system we examined the production of a human glucose transceptor and 11 nutrient transporters and transceptors from S. cerevisiae that have not previously been overexpressed in S. cerevisiae and purified. Whole-cell GFP-fluorescence showed that induction of GFP-fusion synthesis from a galactose-inducible promoter at 15°C resulted in stable accumulation of the fusions in the plasma membrane and in intracellular membranes. Expression levels of the 12 fusions estimated by GFP-fluorescence were in the range of 0.4 mg to 1.7 mg transporter pr. liter cell culture. A detergent screen showed that n-dodecyl-ß-D-maltopyranoside (DDM) is acceptable for solubilization of the membrane-integrated fusions. Extracts of solubilized membranes were prepared with this detergent and used for purifications by Ni-NTA affinity chromatography, which yielded partially purified full-length fusions. Most of the fusions were readily cleaved at a TEV protease site between the membrane protein and the GFP-8His tag. Using the yeast oligopeptide transporter Ptr2 as an example, we further demonstrate that almost pure transporters, free of the GFP-8His tag, can be achieved by TEV protease cleavage followed by reverse immobilized metal-affinity chromatography. The quality of the GFP-fusions was analysed by fluorescence size-exclusion chromatography. Membranes solubilized in DDM resulted in preparations containing aggregated fusions. However, 9 of the fusions solubilized in DDM in presence of cholesteryl hemisuccinate and specific substrates, yielded monodisperse preparations with only minor amounts of aggregated membrane proteins. In conclusion, we developed a new effective S. cerevisiae expression system that may be used for production of high-quality eukaryotic membrane proteins for functional and structural analysis.

AB - To produce large quantities of high quality eukaryotic membrane proteins in Saccharomyces cerevisiae, we modified a high-copy vector to express membrane proteins C-terminally-fused to a Tobacco Etch Virus (TEV) protease detachable Green Fluorescent Protein (GFP)-8His tag, which facilitates localization, quantification, quality control, and purification. Using this expression system we examined the production of a human glucose transceptor and 11 nutrient transporters and transceptors from S. cerevisiae that have not previously been overexpressed in S. cerevisiae and purified. Whole-cell GFP-fluorescence showed that induction of GFP-fusion synthesis from a galactose-inducible promoter at 15°C resulted in stable accumulation of the fusions in the plasma membrane and in intracellular membranes. Expression levels of the 12 fusions estimated by GFP-fluorescence were in the range of 0.4 mg to 1.7 mg transporter pr. liter cell culture. A detergent screen showed that n-dodecyl-ß-D-maltopyranoside (DDM) is acceptable for solubilization of the membrane-integrated fusions. Extracts of solubilized membranes were prepared with this detergent and used for purifications by Ni-NTA affinity chromatography, which yielded partially purified full-length fusions. Most of the fusions were readily cleaved at a TEV protease site between the membrane protein and the GFP-8His tag. Using the yeast oligopeptide transporter Ptr2 as an example, we further demonstrate that almost pure transporters, free of the GFP-8His tag, can be achieved by TEV protease cleavage followed by reverse immobilized metal-affinity chromatography. The quality of the GFP-fusions was analysed by fluorescence size-exclusion chromatography. Membranes solubilized in DDM resulted in preparations containing aggregated fusions. However, 9 of the fusions solubilized in DDM in presence of cholesteryl hemisuccinate and specific substrates, yielded monodisperse preparations with only minor amounts of aggregated membrane proteins. In conclusion, we developed a new effective S. cerevisiae expression system that may be used for production of high-quality eukaryotic membrane proteins for functional and structural analysis.

U2 - 10.1371/journal.pone.0076851

DO - 10.1371/journal.pone.0076851

M3 - Journal article

C2 - 24124599

VL - 8

JO - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 10

M1 - e76851

ER -

ID: 51466924