High Yield Expression of the Human Ether-à-Go-Go Related Gene (HERG) in Saccharomyces Cerevisiae
| Autor: | Per Amstrup Pedersen, Dan A. Klaerke, Peter Scharff-Poulsen, Karen Molbaek |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
congenital
hereditary and neonatal diseases and abnormalities 0303 health sciences biology Saccharomyces cerevisiae hERG Biophysics biology.organism_classification Molecular biology Yeast Potassium channel law.invention 03 medical and health sciences 0302 clinical medicine Plasmid Biochemistry law Complementary DNA Recombinant DNA biology.protein cardiovascular diseases Heterologous expression 030217 neurology & neurosurgery 030304 developmental biology |
| Zdroj: | Biophysical Journal. 106(2) |
| ISSN: | 0006-3495 |
| DOI: | 10.1016/j.bpj.2013.11.802 |
| Popis: | The human Ether-a-go-go related gene (hERG) encodes a voltage-gated potassium channel and represents the molecular correlate of the IKr current, which is one of the potassium currents involved in the repolarizing of the cardiac action potential. Inhibition of hERG prolongs the QT interval in the electrocardiogram and enhances the risk for severe or even fatal arrhythmias. Detection of unintended interactions with hERG is therefore an important issue when compounds are approved for drug development. In the present study we have explored Saccharomyces cerevisae as a host for heterologous expression of the hERG channel. Yeast codon optimized hERG cDNA was used to generate expression plasmids producing the hERG channel C-terminally fused with either a His10 or a TEV-GFP-His8 tag. The latter was generated to ease quantification of the expression level, to allow live cell bioimaging, development of a purification protocol and assessment of the quality of the recombinant protein. Both gene fusions were expressed from a galactose inducible promoter located on a plasmid with a regulatable copy number in a yeast strain overexpressing the Gal4 transcriptional activator. 48 hours after induction recombinant hERG accumulated to approximately 1.6% of total membrane content when produced at 15°C in amino acid complemented media. A solubilization screen established Fos-Choline-12 as a superior detergent for hERG solubilization. Solubilization in Fos-Choline-12 supplemented with cholesterol hemisuccinate generated a monodisperse FSEC (fluorescent size-exclusion chromatography) profile and caused recombinant hERG to elute in its native tetrameric form. In-gel fluorescence of SDS-separated yeast membranes showed that recombinant hERG protein has the expected molecular weight. Complementation assays in S. cerevisiae revealed that the heterologuosly expressed hERG is able to rescue the high potassium requirement of a trk1Δ, trk2Δ yeast strain indicating that the recombinant hERG is functional. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|