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The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications
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 establis...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6764986/ https://www.ncbi.nlm.nih.gov/pubmed/31562305 http://dx.doi.org/10.1038/s41467-019-12301-7 |
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author | Götzke, Hansjörg Kilisch, Markus Martínez-Carranza, Markel Sograte-Idrissi, Shama Rajavel, Abirami Schlichthaerle, Thomas Engels, Niklas Jungmann, Ralf Stenmark, Pål Opazo, Felipe Frey, Steffen |
author_facet | Götzke, Hansjörg Kilisch, Markus Martínez-Carranza, Markel Sograte-Idrissi, Shama Rajavel, Abirami Schlichthaerle, Thomas Engels, Niklas Jungmann, Ralf Stenmark, Pål Opazo, Felipe Frey, Steffen |
author_sort | Götzke, Hansjörg |
collection | PubMed |
description | 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 (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. |
format | Online Article Text |
id | pubmed-6764986 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-67649862019-09-30 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 Opazo, Felipe Frey, Steffen Nat Commun Article 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 (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. Nature Publishing Group UK 2019-09-27 /pmc/articles/PMC6764986/ /pubmed/31562305 http://dx.doi.org/10.1038/s41467-019-12301-7 Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Götzke, Hansjörg Kilisch, Markus Martínez-Carranza, Markel Sograte-Idrissi, Shama Rajavel, Abirami Schlichthaerle, Thomas Engels, Niklas Jungmann, Ralf Stenmark, Pål Opazo, Felipe Frey, Steffen The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications |
title | The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications |
title_full | The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications |
title_fullStr | The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications |
title_full_unstemmed | The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications |
title_short | The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications |
title_sort | alfa-tag is a highly versatile tool for nanobody-based bioscience applications |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6764986/ https://www.ncbi.nlm.nih.gov/pubmed/31562305 http://dx.doi.org/10.1038/s41467-019-12301-7 |
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