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Method to increase the yield of eukaryotic membrane protein expression in Saccharomyces cerevisiae for structural and functional studies.

ABSTRACT: Despite recent successes in the structure determination of eukaryotic membrane proteins, the total number of structures of these important proteins is severely underrepresented in the Protein Data Bank. Although prokaryotic homologues provide valuable mechanistic insight, they often lack crucial details, such as post-translational modification and additional intra or extracellular domains that are important for understanding the function and regulation of these proteins in eukaryotic cells. The production of milligram quantities of recombinant protein is still a serious obstacle to the structural and functional characterization of these proteins. Here, we report a modification to a previously described over expression system using the simple eukaryote Saccharomyces cerevisiae that can increase overall protein yield and improve downstream purification procedures. Using a metabolic marker under the control of a truncated promoter, we show that expression levels for several membrane transporters are increased fourfold. We further demonstrate that the increase in expression for our test proteins resulted in a concomitant increase in functional protein. Using this system, we were able to increase the expression level of a plant transporter, NRT1.1, which was a key factor in its structural and functional characterization.

SUBMITTER: Parker JL

PROVIDER: S-EPMC4230410 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project 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.

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Method to increase the yield of eukaryotic membrane protein expression in Saccharomyces cerevisiae for structural and functional studies.

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