A rationally designed and highly versatile epitope tag for nanobody-based purification, detection and manipulation of proteins
bioRxiv(2019)
摘要
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 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 detection of proteins. By solving the crystal structure of the complex we were able to design a nanobody mutant (NbALFA) 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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关键词
Nanobody,sdAbs,epitope tag,affinity,immunoprecipitation,native elution,super-resolution microscopy,STED,DNA-PAINT,CAR-T,lymphocytes,cell isolation
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