The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications
説明
<jats:title>Abstract</jats:title><jats:p>Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a rationally designed epitope tag that serves a remarkably broad spectrum of applications in life sciences while outperforming established tags like the HA-, FLAG®- or myc-tag. The ALFA-tag forms a small and stable α-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We characterize a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in vivo detection of proteins. We show the crystal structure of the complex that enabled us to design a nanobody mutant (NbALFA<jats:sup>PE</jats:sup>) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.</jats:p>
収録刊行物
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- Nature Communications
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Nature Communications 10 (1), 4403-, 2019-09-27
Springer Science and Business Media LLC
