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Götzke, Hansjörg; Kilisch, Markus; Martinez-Carranza, Markel; Sograte-Idrissi, Shama; Rajavel, Abirami; Schlichthaerle, Thomas; Engels, Niklas; Jungmann, Ralf; Stenmark, Pal; Opazo, Felipe and Frey, Steffen (2019): The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. In: Nature Communications, Vol. 10, 4403 [PDF, 2MB]


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 (R)- or myc-tag. The ALFA-tag forms a small and stable a-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(PE)) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.

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