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 (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.
Item Type: | Journal article |
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Faculties: | Physics |
Subjects: | 500 Science > 530 Physics |
URN: | urn:nbn:de:bvb:19-epub-82638-3 |
ISSN: | 2041-1723 |
Language: | English |
Item ID: | 82638 |
Date Deposited: | 15. Dec 2021, 15:02 |
Last Modified: | 27. Jan 2022, 06:48 |