Saccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography
| Autor: | Per Amstrup Pedersen, Peter Scharff-Poulsen |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Recombinant Fusion Proteins
medicine.medical_treatment Science Detergents Genetic Vectors Green Fluorescent Proteins Saccharomyces cerevisiae Gene Expression Chromatography Affinity Affinity chromatography Gene Order TEV protease medicine Humans Multidisciplinary Protease biology Protein Stability Membrane transport protein Tobacco etch virus Membrane Transport Proteins Biological Transport biology.organism_classification Protein Transport Membrane Membrane protein Biochemistry Proteolysis biology.protein Medicine Research Article |
| Zdroj: | PLoS ONE, Vol 8, Iss 10, p e76851 (2013) 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, vol. 8, no. 10, e76851 . https://doi.org/10.1371/journal.pone.0076851 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 ', P L o S One, vol. 8, no. 10, e76851 . https://doi.org/10.1371/journal.pone.0076851 PLoS ONE |
| ISSN: | 1932-6203 |
| Popis: | To produce large quantities of high quality eukaryotic membrane proteins in Saccharomyces cerevisiae, we modified a highcopy 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 GFPfluorescenceshowed that induction of GFP-fusion synthesis from a galactose-inducible promoter at 15uC resulted in stable accumulation of the fusions in the plasma membrane and in intracellular membranes. Expression levels of the 12 fusionsestimated 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 anexample, 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. |
| Databáze: | OpenAIRE |
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