2. Academic Validation
  3. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications

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

  • Nat Commun. 2019 Sep 27;10(1):4403. doi: 10.1038/s41467-019-12301-7.
Hansjörg Götzke 1,  Markus Kilisch 1 2,  Markel Martínez-Carranza 3,  Shama Sograte-Idrissi 4 5,  Abirami Rajavel 1,  Thomas Schlichthaerle 6 7,  Niklas Engels 8,  Ralf Jungmann 6 7,  Pål Stenmark 3 9,  Felipe Opazo 10 11 12,  Steffen Frey 13
Affiliations

Affiliations

  • 1 NanoTag Biotechnologies GmbH, Rudolf-Wissell-Straße 28a, 37079, Göttingen, Germany.
  • 2 Institute of Molecular Biology, University Medical Center Göttingen, Humboldtallee 23, 37073, Göttingen, Germany.
  • 3 Department of Biochemistry and Biophysics, Stockholm University, S-106 91, Stockholm, Sweden.
  • 4 Institute of Neuro- and Sensory Physiology, University Medical Center Göttingen, Humboldtallee 23, 37073, Göttingen, Germany.
  • 5 Center for Biostructural Imaging of Neurodegeneration (BIN), University Medical Center Göttingen, Von-Siebold-Straße 3a, 37075, Göttingen, Germany.
  • 6 Faculty of Physics and Center for Nanoscience, LMU Munich, Geschwister-Scholl-Platz 1, 80539, Munich, Germany.
  • 7 Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152, Martinsried, Germany.
  • 8 Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Humboldtallee 34, 37073, Göttingen, Germany.
  • 9 Department of Experimental Medical Science, Lund University, Lund, 221 00, Sweden.
  • 10 NanoTag Biotechnologies GmbH, Rudolf-Wissell-Straße 28a, 37079, Göttingen, Germany. fopazo@gwdg.de.
  • 11 Institute of Neuro- and Sensory Physiology, University Medical Center Göttingen, Humboldtallee 23, 37073, Göttingen, Germany. fopazo@gwdg.de.
  • 12 Center for Biostructural Imaging of Neurodegeneration (BIN), University Medical Center Göttingen, Von-Siebold-Straße 3a, 37075, Göttingen, Germany. fopazo@gwdg.de.
  • 13 NanoTag Biotechnologies GmbH, Rudolf-Wissell-Straße 28a, 37079, Göttingen, Germany. steffen.frey@nano-tag.com.
PMID: 31562305 DOI: 10.1038/s41467-019-12301-7
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.

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