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

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 (NbALFAPE) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.

3T3 cells were transiently transfected with EGFP fusions harboring N-or C-terminal ALFA tags. Non-tagged EGFP from pEGFP-N1 served as a control. The localization of the respective EGFP variants was analyzed on 6-7 individual images for each construct. Between 120 and 130 cells were imaged per construct and the localization of EGFP was analyzed. In general, each EGFP construct displayed a distribution across the cytosol and the nucleus. Cells were distributed into three groups (slightly nuclear, uniform and other) according to the observed nucleocytoplasmic localization of EGFP. Standard deviations represented by error bars were calculated from values obtained from individual images. Differences between the localizations of tagged and non-tagged EGFP variants were statistically not significant (Student's ttest). COS-7 cells were co-transfected with an NbALFA-mScarlet-I fusion and indicated ALFAtagged target proteins. Target proteins were detected either using the intrinsic EGFP fluorescence (TOM70-EGFP-ALFA) or by immunofluorescence using NbALFA-Ab635P (ALFA-Vimentin). In parallel, NbALFA-mScarlet-I was detected by its red fluorescence. The excellent co-localization shows that NbALFA expressed in the cytoplasm of mammalian cells can be used for targeting ALFA-tagged proteins in living cells. Scale bars: 20µm. Colors scheme used for sketches: NbALFA (orange), ALFA-tag (blue), GFP (green), mScarlet-I (red), FLAG tag (grey), vimentin (light blue). The lower panel recapitulates data shown in Figure 1g and is repeated here for a better comparison.  Lines represent fits to a single exponential. Half times are given for peptide elution from AL-FA Selector PE only. For all substrate proteins, peptide elution from ALFA Selector ST was inefficient even after prolonged incubation. In the absence of ALFA peptide, the ALFA-tagged target proteins remained tightly bound to both resins. Note that peptide elution of shGFP2 harboring an N-terminal ALFA tag from ALFA Selector PE is quicker as compared to substrates harboring the ALFA tag at internal or C-terminal positions. Panel (a) recapitulates data shown in Figure 4b and is repeated here to allow a direct comparison. Colors scheme used for sketches: NbALFA (orange), ALFA-tag (blue), GFP (green), bdSUMO (grey).

Supplementary Figure 8 -related to Figure 5c: Pull-down of a native E. coli YfgM-PpiD inner membrane protein complex using the ALFA Selector (complete blots).
A yfgM deletion strain was complemented with either C-terminally ALFA-tagged (left panel) or untagged YfgM (right panel; control reaction) expressed from a low-copy vector. Membrane protein complexes were solubilized from total lysate using DDM. Complexes containing YfgM-ALFA were purified in a single step using ALFA Selector PE affinity resin and eluted under native conditions using 200µM ALFA peptide. Samples corresponding to 1/800 of the input and non-bound material or 1/80 of eluate fractions were resolved by SDS-PAGE and analyzed by Western blot. A rabbit serum raised against the YfgM-PpiD complex recognized both, PpiD and YfgM, in the input fractions. ALFA Selector PE specifically immunoprecipitated the native protein complex comprising ALFA-tagged YfgM and its interaction partner PpiD. In the control reaction (no ALFA tag on YfgM), both proteins were absent in the eluate. The figure recapitulates data shown in Figure 5c, here, however, complete membranes are shown. Asterisks mark non-specific bands recognized by the polyclonal antiserum.
Supplementary Figure 9 -related to Figure 6: Gating strategy to identify lymphocytes.
Total human PBMCs were left untreated (Before sorting) or isolated using an ALFA Selector PE resin loaded with an ALFA-tagged anti-human CD62L nanobody (After sorting) as shown in Figure 6. Forward-scatter/side-scatter plots showing the lymphocyte gate (black oval) that has been used to exclude cellular debris from the analysis. The numbers give the percentage of all events in the lymphocyte gate. Identical gates have been applied to all analyses shown in Figure 6. - Supplementary Table 3. List of intermolecular interactions between NbALFA and the ALFA peptide.

Properties of binder
(a) ALFA peptide residues are marked by an apostrophe.