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bioRxiv · 10.1101/640771

A rationally designed and highly versatile epitope tag for nanobody-based purification, detection and manipulation of proteins

Goetzke, H.·Kilisch, M.·Martinez-Carranza, M.·Sograte-Idrissi, S.·Rajavel, A.·Schlichthaerle, T.·Engels, N.·Jungmann, R.·Stenmark, P.·Opazo, F.·Frey, S.
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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 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 -helix that is functional irrespective of its position on the target protein 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.

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Goetzke, H., Kilisch, M., Martinez-Carranza, M., Sograte-Idrissi, S., Rajavel, A., Schlichthaerle, T., Engels, N., Jungmann, R., Stenmark, P., Opazo, F., Frey, S.. 2019-05-17. A rationally designed and highly versatile epitope tag for nanobody-based purification, detection and manipulation of proteins. https://doi.org/10.1101/640771

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