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

Author

Listed:
  • Hansjörg Götzke

    (NanoTag Biotechnologies GmbH)

  • Markus Kilisch

    (NanoTag Biotechnologies GmbH
    University Medical Center Göttingen)

  • Markel Martínez-Carranza

    (Stockholm University)

  • Shama Sograte-Idrissi

    (University Medical Center Göttingen
    University Medical Center Göttingen)

  • Abirami Rajavel

    (NanoTag Biotechnologies GmbH)

  • Thomas Schlichthaerle

    (Faculty of Physics and Center for Nanoscience, LMU Munich
    Max Planck Institute of Biochemistry)

  • Niklas Engels

    (University Medical Center Göttingen)

  • Ralf Jungmann

    (Faculty of Physics and Center for Nanoscience, LMU Munich
    Max Planck Institute of Biochemistry)

  • Pål Stenmark

    (Stockholm University
    Department of Experimental Medical Science, Lund University)

  • Felipe Opazo

    (NanoTag Biotechnologies GmbH
    University Medical Center Göttingen
    University Medical Center Göttingen)

  • Steffen Frey

    (NanoTag Biotechnologies GmbH)

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.

Suggested Citation

  • Hansjörg Götzke & Markus Kilisch & Markel Martínez-Carranza & Shama Sograte-Idrissi & Abirami Rajavel & Thomas Schlichthaerle & Niklas Engels & Ralf Jungmann & Pål Stenmark & Felipe Opazo & Steffen Fr, 2019. "The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications," Nature Communications, Nature, vol. 10(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-12301-7
    DOI: 10.1038/s41467-019-12301-7
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    Cited by:

    1. Xiansha Xiao & Allison Fay & Pablo Santos Molina & Amanda Kovach & Michael S. Glickman & Huilin Li, 2024. "Structure of the M. tuberculosis DnaK−GrpE complex reveals how key DnaK roles are controlled," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    2. Irina Shlosman & Elayne M. Fivenson & Morgan S. A. Gilman & Tyler A. Sisley & Suzanne Walker & Thomas G. Bernhardt & Andrew C. Kruse & Joseph J. Loparo, 2023. "Allosteric activation of cell wall synthesis during bacterial growth," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    3. Kevin Wu & Samuel Itskanov & Diane L. Lynch & Yuanyuan Chen & Aasha Turner & James C. Gumbart & Eunyong Park, 2024. "Substrate recognition mechanism of the endoplasmic reticulum-associated ubiquitin ligase Doa10," Nature Communications, Nature, vol. 15(1), pages 1-18, December.
    4. Leo Kiss & Tyler Rhinesmith & Jakub Luptak & Claire F. Dickson & Jonas Weidenhausen & Shannon Smyly & Ji-Chun Yang & Sarah L. Maslen & Irmgard Sinning & David Neuhaus & Dean Clift & Leo C. James, 2023. "Trim-Away ubiquitinates and degrades lysine-less and N-terminally acetylated substrates," Nature Communications, Nature, vol. 14(1), pages 1-17, December.
    5. Xudong Chen & Min Xie & Sensen Zhang & Marta Monguió-Tortajada & Jian Yin & Chang Liu & Youqi Zhang & Maeva Delacrétaz & Mingyue Song & Yixue Wang & Lin Dong & Qiang Ding & Boda Zhou & Xiaolin Tian & , 2023. "Structural basis for recruitment of TASL by SLC15A4 in human endolysosomal TLR signaling," Nature Communications, Nature, vol. 14(1), pages 1-13, December.

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