A rationally designed and highly versatile epitope tag for nanobody-based purification, detection and manipulation of proteins
Hansjörg Götzke, Markus Kilisch, Markel Martínez-Carranza, Shama Sograte- Idrissi, Abirami Rajavel, Thomas Schlichthaerle, Niklas Engels, Ralf Jungmann, Pål Stenmark, Felipe Opazo and Steffen Frey
Received date 3rd May 19
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 novel, rationally designed epitope tag that serves an exceptionally broad spectrum of applications in life sciences while outperforming established tags like the HA, FLAG or myc tags. The ALFA-tag forms a small and stable α-helix that is functional irrespective of its position on the targetprotein in prokaryotic and eukaryotic hosts. We developed 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. By solving the crystal structure of the complex we were able 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.
Read in full at bioRxiv.
This is an abstract of a preprint hosted on an independent third party site. It has not been peer reviewed but is currently under consideration at Nature Communications.