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A rationally designed and highly versatile epitope tag for nanobody-based purification, detection and manipulation of proteins

By Hansjörg Götzke, Markus Kilisch, Markel Martínez-Carranza, Shama Sograte-Idrissi, Abirami Rajavel, Thomas Schlichthaerle, Niklas Engels, Ralf Jungmann, Pål Stenmark, Felipe Opazo, Steffen Frey

Posted 17 May 2019
bioRxiv DOI: 10.1101/640771 (published DOI: 10.1038/s41467-019-12301-7)

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 novel, rationally designed epitope tag that serves an exceptionally broad spectrum of applications in life sciences while outperforming established tags like the HA, FLAG or myc tags. 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 developed 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. By solving the crystal structure of the complex we were able to design a nanobody mutant (NbALFAPE) 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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