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
  • 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. [email protected].
  • 11. Institute of Neuro- and Sensory Physiology, University Medical Center Göttingen, Humboldtallee 23, 37073, Göttingen, Germany. [email protected].
  • 12. Center for Biostructural Imaging of Neurodegeneration (BIN), University Medical Center Göttingen, Von-Siebold-Straße 3a, 37075, Göttingen, Germany. [email protected].
  • 13. NanoTag Biotechnologies GmbH, Rudolf-Wissell-Straße 28a, 37079, Göttingen, Germany. [email protected].
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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