The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications

TLDR

Epitope tags are widely used for protein detection, manipulation, and purification, yet their versatility is often limited. The study introduces the ALFA‑tag, a rationally designed epitope tag that offers broad applications and outperforms established tags such as HA, FLAG, and myc. The ALFA‑tag is a small, stable α‑helix that functions regardless of its position in prokaryotic or eukaryotic proteins, and a nanobody (NbALFA) binds it with picomolar affinity; structural insights guided the creation of a mutant nanobody (NbALFA PE) that allows one‑step purification of native ALFA‑tagged proteins and cells via peptide elution. The ALFA‑tag and its nanobody enable super‑resolution microscopy, immunoprecipitation, Western blotting, in‑vivo protein detection, and efficient one‑step purification of native proteins, complexes, and living cells under physiological conditions.

Abstract

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®- 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.