| Naslov: | High yield purification of full-length functional hERG K+ channels produced in Saccharomyces cerevisiae |
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| Avtorji: | ID Molbaek, Karen (Avtor)
ID Hélix-Nielsen, Claus (Avtor) |
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| Datoteke: |  Microbial_Cell_Factories_2015_Molbaek_et_al._High_yield_purification_of_full-length_functional_hERG_K_channels_produced_in_Saccharomyces.pdf (2,85 MB)
MD5: 6F26570564F332C5245E46E5F85CAD46
 http://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-015-0193-9
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| Jezik: | Angleški jezik |
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| Vrsta gradiva: | Znanstveno delo |
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| Tipologija: | 1.01 - Izvirni znanstveni članek |
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| Organizacija: | FKKT - Fakulteta za kemijo in kemijsko tehnologijo
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| Opis: | The hERG potassium channel is essential for repolarization of the cardiac action potential. Due to this vital function, absence of unintended and potentially life-threatening interactions with hERG is required for approval of new drugs. The structure of hERG is therefore one of the most sought-after. To provide purified hERG for structural studies and new hERG biomimetic platforms for detection of undesirable interactions, we have developed a hERG expression platform generating unprecedented amounts of purified and functional hERG channels. Full-length hERG, with or without a C-terminally fused green fluorescent protein (GFP) His 8-tag was produced from a codon-optimized hERG cDNA in Saccharomyces cerevisiae. Both constructs complemented the high potassium requirement of a knock-out Saccharomyces cerevisiae strain, indicating correct tetramer assembly in vivo. Functionality was further demonstrated by Astemizole binding to membrane embedded hERG-GFP-His 8 with a stoichiometry corresponding to tetramer assembly. The 156 kDa hERG-GFP protein accumulated to a membrane density of 1.6%. Fluorescence size exclusion chromatography of hERG-GFP-His 8 solubilized in Fos-Choline-12 supplemented with cholesteryl-hemisuccinate and Astemizole resulted in a monodisperse elution profile demonstrating a high quality of the hERG channels. hERG-GFP-His 8 purified by Ni-affinity chromatography maintained the ability to bind Astemizole with the correct stoichiometry indicating that the native, tetrameric structure was preserved. To our knowledge this is the first reported high-yield production and purification of full length, tetrameric and functional hERG. This significant breakthrough will be paramount in obtaining hERG crystal structures, and in establishment of new high-throughput hERG drug safety screening assays. |
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| Ključne besede: | potassium channel, membrane protein production, functional expression, cardiac action potential |
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| Status publikacije: | Objavljeno |
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| Verzija publikacije: | Objavljena publikacija |
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| Leto izida: | 2015 |
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| Št. strani: | str. 1-16 |
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| Številčenje: | Letn. 14 |
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| PID: | 20.500.12556/DKUM-60151  |
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| ISSN: | 1475-2859 |
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| UDK: | 54 |
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| COBISS.SI-ID: | 18539798 |
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| DOI: | 10.1186/s12934-015-0193-9 |
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| ISSN pri članku: | 1475-2859 |
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| NUK URN: | URN:SI:UM:DK:QGTGZ6VQ |
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| Datum objave v DKUM: | 29.06.2017 |
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| Število ogledov: | 1738 |
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| Število prenosov: | 384 |
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| Metapodatki: |    |
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| Področja: | Ostalo
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