Spot-Tag: a Nanobody-based Peptide-Tag System for Protein Detection, Purification and Imaging Michael Metterlein 1 , Larisa Yurlova 1 , Andrea Buchfellner 1 , Benjamin Ruf 1 , Verena Leingärtner 1 , Jacqueline Bogner 1 , Tina Romer 1 , Philipp D. Kaiser 2 , Felix Hartlepp 1 , Christian Linke-Winnebeck 1* 1 ChromoTek GmbH, Planegg-Martinsried, Germany, 2 Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany * Email to: [email protected] Acknowledgements: This work was supported by a grant “Zentrales Innovationsprogramm Mittelstand“ (ZIM) from the Federal Ministry for Economic Affairs and Energy of Germany. Also, we thank Andreas Thomae at the Core Facility Bioimaging of the Biomedical Center, LMU Munich, for recording confocal and STED images. References: [1] Tränkle, B. et al.: Monitoring interactions and dynamics of endogenous β-catenin with intracellular nanobodies in living cells. Molecular & Cellular Proteomics 14(3), pp. 707-723 (2015). [2] Braun, M.B. et al.: Peptides in headlock - a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy. Scientific reports 6, 19211 (2016). [3] Virant, D. et al.: A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging. Nature Communications 9(1) 930 (2018). Disclaimer: For research use only. ChromoTek`s products and technology are covered by granted patents and pending applications owned by or available to ChromoTek GmbH by exclusive license. ChromoTek, Chromobody, Spot-Tag and Spot-Trap are registered trademarks of ChromoTek GmbH. Nanobody is a registered trademark of Ablynx nv. We have developed a new epitope tag system based on a V H H (also called nanobody®), i.e. a small and highly stable alpaca single-domain antibody. This V H H binds with high affinity to the Spot-Tag®, an engineered 12-amino acid sequence (PDRVRAVSHWSS) derived from a linear epitope within β-catenin. Owing to the unique properties of the anti-Spot-Tag-V H H, the Spot-Tag capture and detection system is universally applicable. The Spot-Tag The anti-Spot nanobody Applications of the Spot-Tag system Immunisation of alpacas with β-catenin yields nanobody that binds unstructured N-terminus [1] (amino acids 16-27). Further screening of 30 variants identifies tag sequence optimised for high affinity: the Spot-Tag. 100 200 300 400 0.0 0.2 0.4 0.6 0.8 Time (s) Binding (nm) 100 200 300 400 0.0 0.2 0.4 0.6 0.8 Time (s) 5 nM 10 nM 15 nM 25 nM 50 nM Origin of the Spot-Tag High affinity of the Spot-Tag → Engineering of original epitope leads to significantly decreased dissociation and thus improved affinity for Spot-Tag. → High affinity binding of Spot-Tag to anti-Spot nanobody K D k on (M -1 s -1 ) k off (s -1 ) β-cat. 16-27 C-term. 45 nM 1.5 ×10 5 6.7 ×10 -3 β-cat. 16-27 N-term. 56 nM 1.4 ×10 5 8.0 ×10 -3 Spot-Tag C-term. 7 nM 1.8 ×10 5 1.3 ×10 -3 Spot-Tag N-term. 6 nM 1.3 ×10 5 7.4 ×10 -4 β-cat.-mCherry Spot-Tag-mCherry 0 2 4 6 8 1.20 1.25 1.30 Urea [M] ratio F(350nm/330nm) 20 40 60 80 1.10 1.15 1.20 1.25 Temperature (°C) Ratio F(350 nm/330 nm) 20 40 60 80 Temperature (°C) 20 40 60 80 0.05 0.10 0.15 Temperature (°C) Scattering (AU) Crystal structure of the anti-Spot nanobody. [2] Characteristics High stability First derivative • Single peptide of 131 amino acids • Small size: 14.7 kDa • Binds Spot-Tag with high affinity • Thorough biochemical and structural characterisation • Recombinant production and thus high batch-to-batch consistency • Binds Spot-Tag fused to N- or C-terminus of a protein • Easily derivatised using fluorophores or agarose matrices Western blot ELISA Immunofluorescence microscopy using Spot-Label Purification and immunoprecipitation using Spot-Trap® A bivalent format of anti-Spot nanobody conjugated to fluorophores (called Spot-Label), allows the imaging of cellular proteins and structures using fluorescence microscopy. → Small size of Spot-Label leads to better tissue penetration. → Spot-Label is the first detection tool directed against a small peptide tag that is applicable to super resolution microscopy studies (STED and also STORM [3] ) owing to minimal label displacement. Spot-Label ATTO594 confocal microscopy imaging of Tom70-GFP- Spot in HeLa cells. Spot-Label ATTO594 STED super resolution imaging of Vimentin- Spot in HeLa cells. GFP-Spot in HEK293T cell lysate 0 50 25 12 6 3 ng: 25 kDa 32 kDa 80 kDa 11 kDa Western blot detection using monovalent Spot-Label ATTO488. For Western blot detection of Spot-Tag proteins, monovalent Spot-Label conjugated to a fluorophore or in conjuction with a secondary antibody can be used. → Spot-Label ATTO488 allows sensitive one-step Western blot detection with minimal background. → Alternatively, anti-Spot nanobody can be detected using anti-llama HPR antibody. -9 -8 -7 -6 -5 0.0 0.5 1.0 M Absorption 450 nm mCherry control mCherry-Spot → Anti-Spot nanobody allows detection and capture of Spot-Tag proteins in ELISA. Coating of mCherry-Spot followed by ELISA analysis using varyiing concentrations of anti-Spot nanobody and anti-llama-HRP. Differential scanning fluorimetry analysis of anti-Spot nanobody. No aggregation observed for anti-Spot nanobody (1 g/l) over a temperature gradient. Chemical stability of anti-Spot nanobody in a chaotrope (urea). The anti-Spot nanobody → has a melting temperature T m of 63 °C. → shows unusually high colloidal stability. → is highly resistant to chaotropes. Spot-Label ATTO594 STED super resolution imaging of Actin- Chromobody-Spot in HeLa cells. Anti-Spot nanobody covalently coupled to agarose beads or magnetic agarose beads (called Spot-Trap) enables the immunoprecipitation and purification of Spot-Tag fusion proteins. → High affinity allows the purification of low- abundance proteins. → Bound protein can be eluted using free Spot peptide. → High chemical stability leads to extraordinary chemical compatibility of Spot-Trap → Chemical stability also permits repeated use of Spot-Trap Input Flow-through Elution 80 kDa 25 kDa 32 kDa 11 kDa Input Flow-through Elution fractions 80 kDa 25 kDa 32 kDa 11 kDa Spot-Trap immunopre- cipitation of GFP-Spot from HEK 293T lysate. Spot-Trap affinity purification of GFP-Spot from HEK 293T lysate followed by step-wise Spot peptide (100 μM) elution. 1 M NaCl 2 M NaCl 0.5 % IGEPAL CA-630 1 % IGEPAL CA-630 2 % IGEPAL CA-630 0.1 % SDS 0.1 % Triton X-100 0.5 % Triton X-100 1 % Triton X-100 1 M urea 2 M urea 4 M urea 6 M urea 8 M urea 0.2 % SDS Control Control 1 mM DTT 5 mM DTT 10 mM DTT mCherry-Spot Buffer compatibility test for the Spot-Trap. After precipitation of mCherry-Spot from E. coli, Spot-Trap was washed using control buffer or buffer supplemented with indicated additives. Glutathione cellulose (suppl. 1) α-c-Myc 9E1 (supplier 1) α-c-Myc 9E1 (supplier 2) α-FLAG (supplier 3) Dynabeads (supplier 2) Spot-Trap (magn. agarose) Spot-Trap (agarose) 80 kDa 25 kDa 32 kDa 11 kDa Background of different affinity media in immunoprecipitation from HEK 293T lysate without cognate antigen present. Input Flow-through 1-5 Elution 1-5 Test of long-term stability of Spot-Trap. Spot-Trap was subjected to five full cycles of affinity purification (loading, washing, peptide elution, regeneration) of mCherry-Spot from E. coli. Analysis using Western blotting. Conclusion The Spot-Tag system combines the high affinity and specificity of an antibody-epitope tag system with the stability and small size of an alpaca nanobody. This results in a universal tag-system that will simplify the purification and concurrent analysis of target proteins. Nanobody N C β-catenin aa16-27 Invariable positions (tag:nanobody interactions) Screened and mutated positions Spot-Tag: PD R VR A V S H W SS Conventional antibody Alpaca heavy chain antibody Nanobody/ V H H V H V L C L C H 1 C H 2 C H 3 V H H Spot-Trap® Purification Immunoprecipitation Spot-Label, bivalent Fluorescence microscopy Super resolution microscopy Spot-Label, monov. Western blot anti-Spot V H H Western blot ELISA Alpaca Biolayer interferometry (BLI) kinetics analysis of binding of Spot-Tag nanobody to mCherry tagged with β-catenin 16-27 or Spot-Tag.