The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications

Hansjörg Götzke, Markus Kilisch, Markel Martínez-Carranza, Shama Sograte-Idrissi, Abirami Rajavel, Thomas Schlichthaerle, Niklas Engels, Ralf Jungmann, Pål Stenmark, Felipe Opazo () and Steffen Frey ()
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Hansjörg Götzke: NanoTag Biotechnologies GmbH
Markus Kilisch: NanoTag Biotechnologies GmbH
Markel Martínez-Carranza: Stockholm University
Shama Sograte-Idrissi: University Medical Center Göttingen
Abirami Rajavel: NanoTag Biotechnologies GmbH
Thomas Schlichthaerle: Faculty of Physics and Center for Nanoscience, LMU Munich
Niklas Engels: University Medical Center Göttingen
Ralf Jungmann: Faculty of Physics and Center for Nanoscience, LMU Munich
Pål Stenmark: Stockholm University
Felipe Opazo: NanoTag Biotechnologies GmbH
Steffen Frey: NanoTag Biotechnologies GmbH

Nature Communications, 2019, vol. 10, issue 1, 1-12

Abstract: Abstract 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 (NbALFAPE) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.

Date: 2019
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DOI: 10.1038/s41467-019-12301-7

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