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The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications
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Journal Article
The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications
2019
Overview
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.
Epitope tags are widely used in various applications, but often lack versatility. Here, the authors introduce a small, alpha helical tag, which is recognized by a high affinity nanobody and can be used in a range of different applications, from protein purification to super-resolution imaging and in vivo detection of proteins.
Publisher
Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
Subject
/ 13
/ 13/1
/ 13/31
/ 13/44
/ 13/51
/ 14
/ 14/1
/ 14/10
/ 14/63
/ 82/1
/ 82/16
/ 82/51
/ 82/83
/ 9/10
/ Animals
/ biokemi
/ Elution
/ Epitopes
/ Green Fluorescent Proteins - genetics
/ Green Fluorescent Proteins - metabolism
/ Humanities and Social Sciences
/ Humans
/ Mice
/ Mutation
/ Proteins
/ Recombinant Fusion Proteins - genetics
/ Recombinant Fusion Proteins - metabolism
/ Science
/ Single-Domain Antibodies - chemistry
/ Single-Domain Antibodies - genetics
/ Single-Domain Antibodies - metabolism
/ Tags
