RT Journal Article SR Electronic T1 A rationally designed and highly versatile epitope tag for nanobody-based purification, detection and manipulation of proteins JF bioRxiv FD Cold Spring Harbor Laboratory SP 640771 DO 10.1101/640771 A1 Hansjörg Götzke A1 Markus Kilisch A1 Markel Martínez-Carranza A1 Shama Sograte-Idrissi A1 Abirami Rajavel A1 Thomas Schlichthaerle A1 Niklas Engels A1 Ralf Jungmann A1 Pål Stenmark A1 Felipe Opazo A1 Steffen Frey YR 2019 UL http://biorxiv.org/content/early/2019/06/07/640771.abstract AB 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.