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

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 ALFAtag 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 in-vivo detection of proteins. By solving the crystal structure of the complex we were able 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.

Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

“…Although larger proteins can enter cilia by active transport processes, this size cutoff may limit the size of proteins that can be targeted to cilia by a piggyback mechanism using a cilia-localized nanobody. Importantly, a recently developed nanobody, targeting a small alpha-helical epitope tag of 13 amino acids, NbALFA, also works in the cytosol and may reduce the overall size of the protein complexes targeted to primary cilia (Götzke, Kilisch et al, 2019).…”

A nanobody that recognizes a 14-residue peptide epitope in the E2 ubiquitin-conjugating enzyme UBC6e modulates its activity

“…There are few reported VHHspeptide pairs that engage in high affinity interactions. Notable examples include the NbSyn2-EPEA system from an α-synuclein-binding VHH 25 , the BC2-Spot Tag system from a betacatenin binding VHH 26,27 , and the newly reported Alfa tag system from a VHH raised against a designed α-helical peptide 28 . These VHH-peptide pairs have been used for a variety of applications, ranging from traditional molecular biology approaches such as affinity purifications and immunoprecipitations, to more specialized approaches such as super resolution microscopy and immune cell purification workflows [25][26][27][28] .…”

“…The BC2 nanobody binds a peptide epitope of approximately the same length as VHH05 (12 residues for BC2 vs 14 for VHH05); however, beta-catenin is widely distributed at the cell surface and in the nucleus, potentially complicating some applications 26,27 . The Alfa tag VHH recognizes with very high affinity a 14-mer peptide not found in common organisms; however, this tag remains to be tested in diverse settings 28 . Application of the VHH05-peptide interaction reported here should complement the properties and applications of these existing systems.…”

“…The immunoprecipitation by VHH05 was slightly more efficient than that achieved by a VHH that recognizes an epitope tag from beta-catenin (VHHBC2) and the correspondingly tagged eGFP 26,27 . A VHH reported to recognize a 4-residue fragment from α-synuclein (NbSyn2, named VHHEPEA here) 25,28 failed to immunoprecipitate under these conditions. These experiments unambiguously map the epitope recognized by VHH05 to the segment composed of residues 170-184, with binding independent of the remainder of UBC6e.…”

Reflections on the use of protein binders to study protein function in developmental biology

“…Therefore, the use of recently reported intrabodies against small tags, such as SunTag (19aa) Degraded in absence of the antigen (GFP) (Tang et al, 2016) Lysine-less VHH4 (GFP, YFP, Venus) VHH4 in which lysines have been substituted for Arginines (Daniel et al, 2018) mCherry VHH (Fridy et al, 2014) MoonTag (gp41, 15aa linear epitope) (Boersma et al, 2019) BC2 VHH (BC2 tag, 12aa) Not tested as intrabody. (Braun et al, 2016) NbALFA (ALFAtag, 15aa) (Götzke et al, 2019) Darpin 3G86.32 Anti-GFP DARPin (GFP) (Brauchle et al, 2014) 2 m22 Anti-mCherry DARPin (mCherry) (Brauchle et al, 2014) E11 and G01 Anti-TFP DARPins (TFP) (Vigano et al, 2018) scFv HA frankenbody (HA, 9aa linear epitope) (Zhao et al, 2018) Suntag (GCN4 v4, 19aa linear epitope) Aggregation at high levels unless fused to sfGFP-GB1 scaffold (Tanenbaum, Gilbert, Qi, Weissman, & Vale, 2014) BGP7 ScFv (BGP7, 7aa linear epitope) (Lim, Ichinose, Shinoda, & Ueda, 2007;Wongso, Dong, Ueda, & Kitaguchi, 2017) (15aa), promises to be a great addition to the protein binder toolbox (Boersma et al, 2019;Gotzke et al, 2019;Tanenbaum et al, 2014;Zhao et al, 2018). In most cases, the short peptide tags recognized by these intrabodies has been used in multimerized form such as to recognize single protein particles; whether these short tags also work well when integrated as a single tag remains to be tested.…”