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

Götzke, Hansjörg ; Kilisch, Markus ; Martínez-Carranza, Markel ; Sograte-Idrissi, Shama ; Rajavel, Abirami ; Schlichthaerle, Thomas ; Engels, Niklas ; Jungmann, Ralf ; Stenmark, Pål LU orcid and Opazo, Felipe , et al. (2019) In Nature Communications 10.
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... (More)

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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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Nature Communications
volume
10
article number
4403
publisher
Nature Publishing Group
external identifiers
  • pmid:31562305
  • scopus:85072701941
ISSN
2041-1723
DOI
10.1038/s41467-019-12301-7
language
English
LU publication?
yes
id
36d036e0-e8e9-47c0-a056-c30beca2a519
date added to LUP
2019-10-02 07:40:47
date last changed
2024-07-11 05:43:23
@article{36d036e0-e8e9-47c0-a056-c30beca2a519,
  abstract     = {{<p>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.</p>}},
  author       = {{Götzke, Hansjörg and Kilisch, Markus and Martínez-Carranza, Markel and Sograte-Idrissi, Shama and Rajavel, Abirami and Schlichthaerle, Thomas and Engels, Niklas and Jungmann, Ralf and Stenmark, Pål and Opazo, Felipe and Frey, Steffen}},
  issn         = {{2041-1723}},
  language     = {{eng}},
  month        = {{09}},
  publisher    = {{Nature Publishing Group}},
  series       = {{Nature Communications}},
  title        = {{The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications}},
  url          = {{http://dx.doi.org/10.1038/s41467-019-12301-7}},
  doi          = {{10.1038/s41467-019-12301-7}},
  volume       = {{10}},
  year         = {{2019}},
}