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The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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Number of Authors: 112019 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 4403Article in journal (Refereed) Published
Abstract [en]

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 (R)- or myc-tag. The ALFA-tag forms a small and stable a-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 (NbALFA(PE)) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.

Place, publisher, year, edition, pages
2019. Vol. 10, article id 4403
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-175701DOI: 10.1038/s41467-019-12301-7ISI: 000488232600011PubMedID: 31562305OAI: oai:DiVA.org:su-175701DiVA, id: diva2:1369975
Available from: 2019-11-13 Created: 2019-11-13 Last updated: 2020-01-29Bibliographically approved
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Martínez-Carranza, MarkelRajavel, AbiramiStenmark, PålOpazo, Felipe
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