Saccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography.

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 localizat...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Peter Scharff-Poulsen, Per Amstrup Pedersen
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/df5370a61a6c4eb19a9c18f6ebc65fb6
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:df5370a61a6c4eb19a9c18f6ebc65fb6
record_format	dspace
spelling	oai:doaj.org-article:df5370a61a6c4eb19a9c18f6ebc65fb62021-11-18T08:52:20ZSaccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography.1932-620310.1371/journal.pone.0076851https://doaj.org/article/df5370a61a6c4eb19a9c18f6ebc65fb62013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24124599/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203To 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.Peter Scharff-PoulsenPer Amstrup PedersenPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 10, p e76851 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Peter Scharff-Poulsen Per Amstrup Pedersen Saccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography.
description	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.
format	article
author	Peter Scharff-Poulsen Per Amstrup Pedersen
author_facet	Peter Scharff-Poulsen Per Amstrup Pedersen
author_sort	Peter Scharff-Poulsen
title	Saccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography.
title_short	Saccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography.
title_full	Saccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography.
title_fullStr	Saccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography.
title_full_unstemmed	Saccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography.
title_sort	saccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/df5370a61a6c4eb19a9c18f6ebc65fb6
work_keys_str_mv	AT peterscharffpoulsen saccharomycescerevisiaebasedplatformforrapidproductionandevaluationofeukaryoticnutrienttransportersandtransceptorsforbiochemicalstudiesandcrystallography AT peramstruppedersen saccharomycescerevisiaebasedplatformforrapidproductionandevaluationofeukaryoticnutrienttransportersandtransceptorsforbiochemicalstudiesandcrystallography
_version_	1718421233468440576

Saccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography.

Ejemplares similares