Analysis of Instability Phenomena at Current Interruption in Vacuum Arc Discharge Compared with Silver or Copper Electrode
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Author list: Kamata N., Mungkung N., Kinoshita H., Yuji T.
Publisher: Nature Research
Publication year: 2019
Journal: Nature Communications (2041-1723)
Volume number: 47
Issue number: 4
Start page: 1774
End page: 1780
Number of pages: 7
ISSN: 2041-1723
eISSN: 2041-1723
Languages: English-Great Britain (EN-GB)
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Abstract
There is an urgent need for affinity reagents that target phospho-modified sites on individual proteins; however, generating such reagents remains a significant challenge. Here, we describe a genetic selection strategy for routine laboratory isolation of phospho-specific designed ankyrin repeat proteins (DARPins) by linking in vivo affinity capture of a phosphorylated target protein with antibiotic resistance of Escherichia coli cells. The assay is validated using an existing panel of DARPins that selectively bind the nonphosphorylated (inactive) form of extracellular signal-regulated kinase 2 (ERK2) or its doubly phosphorylated (active) form (pERK2). We then use the selection to affinity-mature a phospho-specific DARPin without compromising its selectivity for pERK2 over ERK2 and to reprogram the substrate specificity of the same DARPin towards non-cognate ERK2. Collectively, these results establish our genetic selection as a useful and potentially generalizable protein engineering tool for studying phospho-specific binding proteins and customizing their affinity and selectivity. ฉ 2019, The Author(s).
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