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, 10 (1), 4403

The ALFA-tag Is a Highly Versatile Tool for Nanobody-Based Bioscience Applications

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The ALFA-tag Is a Highly Versatile Tool for Nanobody-Based Bioscience Applications

Hansjörg Götzke et al. Nat Commun.

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

Conflict of interest statement

S.F., H.G., F.O., M.M.-C., and P.S. are inventors on a pending European patent application covering the ALFA system and its use. S.F., H.G., and F.O. are shareholders of NanoTag Biotechnologies GmbH. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Nanobody-based detection of ALFA-tagged proteins in immunofluorescence applications. a Sketch of NbALFA bound to the ALFA-tag. Given are ALFA sequences for tagging at various positions. b Sequence of NbALFA. Gray boxes indicate CDRs 1–3 (complementarity determining regions 1–3; AbM definition). c COS-7 cells transfected with Tom70-EGFP-ALFA were fixed with paraformaldehyde (PFA) and stained with NbALFA coupled to AbberiorStar635P (NbALFA-Ab635P). Left to right: NbALFA-Ab635P; intrinsic EGFP signal; overlay incl. DAPI stain; sketch illustrating the detection of Tom70-EGFP-ALFA. Scale bars: 20 µm. d ALFA-vimentin was detected with NbALFA-Ab635P after fixation with 4% PFA, 2% glutaraldehyde (GA), or 100% Methanol (MeOH). Scale bars: 20 µm. e STED and confocal images of COS-7 cell transiently transfected with ALFA-vimentin and stained with NbALFA-Ab635P. Color scheme: Red Hot (ImageJ). Scale bars: 2.5 µm. f HeLa cells transfected with ALFA-vimentin were stained with NbALFA bearing a 10-nucleotide single stranded DNA before imaging by 3D DNA-PAINT. Scale bars: 2.5 µm. The histogram refers to a region (small yellow rectangle) where 2 vimentin filaments are resolved although being only ~90 nm apart. The localization precision was 5.2 nm. g COS-7 cells were co-transfected with an NbALFA-mScarlet-I fusion and ALFA-vimentin. NbALFA-mScarlet-I co-localizes with ALFA-vimentin detected by immunofluorescence using NbALFA-Ab635P. This shows that NbALFA expressed in the cytoplasm of mammalian cells can be used for targeting ALFA-tagged proteins in living cells. Scale bars: 20 µm. N: N-terminus; C: C-terminus, TM: transmembrane domain. Colors scheme used for sketches: NbALFA (orange), ALFA-tag (blue), GFP (green), mScarlet-I (red), fluorophore (red star), FLAG®-tag (gray), vimentin (light blue)
Fig. 2
Fig. 2
Detection of ALFA-tagged target proteins by fluorescent Western blot. a Lysates from COS-7 cells transfected with ALFA-vimentin or a mock control plasmid were analyzed by SDS–PAGE and Western blot. The blot was developed with NbALFA directly coupled to IRDye800CW or a mouse anti-tubulin primary antibody followed by FluoTag-X2 anti-Mouse IRDye680RD. Complete lanes including molecular weight markers are shown in Supplementary Fig. 4a. b Sketch of the E. coli maltose-binding protein (MBP) simultaneously fused to FLAG®-tag (FLAG), HA-tag (HA), myc-tag (myc) and ALFA-tag (ALFA). The multi-tag fusion protein was used for the experiments shown in c, d. ALFA-tag is shown in blue. c Dilution series of the protein sketched in b were spotted onto nitrocellulose membranes. Established monoclonal antibodies (M2, 9E10 and F-7) were used together with a goat anti-mouse secondary antibody coupled IRDye800CW to detect the FLAG®-, myc- and HA-tags, respectively. The ALFA-tag was detected using NbALFA coupled to IRDye800CW. The complete experiment with controls is shown in Supplementary Fig. 4b. d Double-logarithmic plot showing quantification of signals obtained in c in arbitrary units (a.u.) versus the amount of spotted target protein. Lines represent linear fits to the obtained values. Even without signal amplification by a secondary antibody, signals obtained using NbALFA were 3- to > 10-times stronger than by established reagents recognizing the other epitope tags. At the same time, detection with NbALFA was 10-fold more sensitive and showed an excellent linearity over approximately three orders of magnitude
Fig. 3
Fig. 3
Structure of the NbALFA-ALFA peptide complex. a View on the NbALFA-ALFA peptide complex illustrated as cartoon (left) or surface representation (right). NbALFA: orange with CDRs 1–3 colored in yellow. ALFA peptide in blue. For both molecules, the N-terminal is oriented left, the C-terminal right. b Polar interactions within the N-terminal region of the ALFA peptide. ALFA peptide residues are denoted by an apostrophe. S2’ and E5’ form hydrogen bonds with CDR2 (S57, E58, R59 and N61). R3 reaches out to CDR3 and interacts with the backbone of V107 and the side chain of D105. c R3’ and E7’ sandwich F110 on the nanobody, forming a cation-Pi interaction (reviewed in ref. ). d Illustration of a hydrophobic cluster (L4’, L8’ and L12’) facing the nanobody’s hydrophobic cavity. e Polar interaction near the C-terminal of the ALFA peptide. The backbone of E14’ forms hydrogen bonds with R65 while the side chain of R11’ interacts with D112 and Y42 on the five-stranded β-sheet. Interestingly, Y42 has been described as a conserved residue in nanobodies of this particular architecture
Fig. 4
Fig. 4
Immunoprecipitation of ALFA-tagged proteins using ALFA Selector resins. a Sketch of shGFP2-ALFA bound to a resin coupled to NbALFA (orange). b, c ALFA SelectorST or ALFA SelectorPE were charged with shGFP2 harboring a C-terminal ALFA-tag. To estimate off-rates, the resins were incubated with an excess of free ALFA peptide at 25 °C. Control reactions were carried out without peptide. shGFP2 released from the resin was quantified using its fluorescence. The graph b shows mean fluorescence readings, as well as standard deviations (error bars; n = 3). Lines represent fits to a single exponential. A photo was taken upon UV illumination after 3 h of elution (c). d Resistance towards stringent washing steps. Both ALFA Selector variants were charged with either ALFA-shGFP2 or shGFP2-ALFA and incubated with a 10-fold volume of the indicated substances for 1 h at 25 °C with shaking. Without further washing steps, photos were taken upon UV illumination after sedimentation of the beads. A semi-quantitative evaluation of binding strengths is given below the images: Triple plus – very strong, double plus – strong, single plus – weak, minus – not detectable, asterisk – GFP fluorescence partially impaired by SDS. e Resistance towards various pH: Similar to d, here, however, the resins were washed to remove non-bound material after incubating at indicated pH for 30 min. Photos were taken after re-equilibration in PBS to allow for recovery of the GFP fluorescence. Color code used in sketches: NbALFA (orange), shGFP2 (green), ALFA-tag (blue)
Fig. 5
Fig. 5
One-step affinity purifications using ALFA Selector resins. a, b E. coli (a) or HeLa (b) lysates blended with purified ALFA-tagged shGFP2 (a, left lane) were incubated with ALFA SelectorST, ALFA SelectorPE or an analogous resin without immobilized sdAb (Selector Control). After washing with PBS, the resins were incubated with 200 µM ALFA peptide for 20 min (E1–peptide) before eluting remaining proteins with SDS sample buffer (E2–SDS). Indicated fractions were analyzed by SDS–PAGE and Coomassie staining. Eluate fractions correspond to the material eluted from 1 µL of resin. c Native pull-down of an E. coli inner membrane protein complex. Left: Sketch of the target protein complex. Right: Detergent-treated lysates from a ΔyfgM strain complemented with either C-terminally ALFA-tagged (left panel) or untagged YfgM (right) were incubated with ALFA SelectorPE. After washing with PBS, bound proteins were eluted using 200 µM 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. ALFA SelectorPE 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. Complete blots in Supplementary Fig. 8. Color code used in sketch: PpiD (purple), YfgM (pink)
Fig. 6
Fig. 6
Isolation of naive lymphocytes using an ALFA-tagged nanobody recognizing CD62L. Total human PBMCs were left untreated (Before sorting) or isolated using an ALFA SelectorPE resin loaded with an ALFA-tagged anti-human CD62L nanobody (After sorting). a A sketch of the affinity purification strategy. b Cells were stained with an anti-CD62L antibody and analyzed by flow cytometry. c The same cells as in b were stained with antibodies directed against CD3, CD19 and CD62L, and analyzed by flow cytometry. A forward scatter/side scatter gate was set on lymphocytes in all analyses (Supplementary Fig. 9). Color code used in a: NbALFA (orange), ALFA-tag (blue), CD62L (light gray), NbCD62L (dark gray), CD62L+ T-lymphocyte (yellow)

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