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- W2912405238 abstract "Kir1.1a, a product of the KCNJ1 gene, is a plasmalemmal inwardly rectifying potassium channel inhibited by ATP. Kir1.1b is a mitochondrial isoform of Kir1.1 lacking first 19 amino acids. Kir1.1b is believed to be a part of mitoK(ATP) channel. However, the data directly linking the protein to this channel activity is limited. It is known that Kir1.1 protein forms homotetramers. Each subunit is characterized by cytoplasmic N- and C-termini and by a transmembrane part of two helices flanking a P-loop segment. The small amount of Kir1.1 protein in mitochondria of H9c2 cells prompted us to express the Kir1.1 protein in E. coli and purify Kir1.1 channels for further studies. Codon-optimized ORF encoding for Kir1.1 was fused to sequences of several expression tags, which could assist membrane insertion and folding of eukaryotic proteins in bacteria (SUMO, pOmpF and MISTIC). In addition, fusion proteins contained affinity tag for purification (6-His tag). The Kir1.1-6xHis construct was chosen for further studies due to its low degradation and the highest membrane expression level. Several detergents were screened and n-dodecyl β-D-maltoside was used for protein purification (based on solubilization efficiency and tetramer stability). This work was supported by Polish National Science Center, grant no. 2015/19/B/NZ1/02794." @default.
- W2912405238 created "2019-02-21" @default.
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- W2912405238 date "2019-02-01" @default.
- W2912405238 modified "2023-10-18" @default.
- W2912405238 title "Synthesis of Romk1/2 Protein in E.Coli" @default.
- W2912405238 doi "https://doi.org/10.1016/j.bpj.2018.11.1329" @default.
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