20.500.12556/DKUM-60151 High yield purification of full-length functional hERG K+ channels produced in Saccharomyces cerevisiae 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. potassium channel membrane protein production functional expression cardiac action potential kalijev kanalček produkcija membranskih proteinov akcijski potencial srca true false true Angleški jezik Slovenski jezik Znanstveno delo 2016-06-09 03:14:57 2017-06-29 12:37:27 2022-06-22 03:06:04 2019-07-11 11:37:02 2015 0 0 str. 1-16 Letn. 14 2015 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1475-2859 54 2609172 18539798 10.1186/s12934-015-0193-9 1475-2859 URN:SI:UM:DK:QGTGZ6VQ Microbial_Cell_Factories_2015_Molbaek_et_al._High_yield_purification_of_full-length_functional_hERG_K_channels_produced_in_Saccharomyces.pdf Microbial_Cell_Factories_2015_Molbaek_et_al._High_yield_purification_of_full-length_functional_hERG_K_channels_produced_in_Saccharomyces.pdf 1 6F26570564F332C5245E46E5F85CAD46 663551c4664ae2d0342336dda8c8e97ba68e0c6a73f59aea8fb27ba229827c07 528a396d-7c0e-11eb-bb7a-00155d0001ca https://dk.um.si/Dokument.php?lang=slv&id=113344 http://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-015-0193-9 1 https://dk.um.si/Dokument.php?lang=slv&id=93966 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0