Global Sales Review September 2008

1. Global Sales Review September 2008 An Introduction to: SNAP-Tag Technologies Sal Russello, Ph.D. Andreas Brecht, Ph.D.

2. Goals… • Discuss launch of new “Cellular Imaging and Analysis products” • Discuss the core technology – a unique line of multi-functional tags • Discuss the key products &applications – including live cell imaging • Discuss the potential customer base – primarily academic cell biologists • Hear from YOU – Challenges? Opportunities? What do you need from us?

3. The Basics… When, Where, What? • 80+ new Cellular Imaging and Analysis products “go live” December 10 • Based on a line of tagging technologies from Covalys Biosciences • Most of the 80 products were available from Covalys… they have been reformatted and manufactured by NEB and are NO LONGER available by Covalys… • NEB and Covalys announce an exclusive licensing arrangement in Dec • Product launch will occur at the American Society for Cellular Biology • All products are based on a core technology from Covalys Biosciences

4. An Introduction

5. Top Level Features & Benefits of the Technology • Enabling: investigate protein dynamics in live cells • Flexibility: widest range of applications with a single construct • Live cell imaging, fixed cell imaging, protein association, pulldowns, etc… • Covalent: Irreversible with defined stoichiometry

6. “Flagship Technology” – SNAP-tag Mechanism • Derived from O6 Alkylguanine-DNA Alkyltransferase (AGT) • Engineered to interact rapidly with BG-substrates • One SNAP-tag = One precisely localized covalent modification • One-way, suicide reaction – non-enzymatic, NO turnover Protein of interest SNAP-tag Benzylguanine Any type of label, or surface

7. Available Tags and Substrate Chemistries SNAP-tag (app 20kDa) CLIP-tag (app 20kDa) ACP-, MCP-tag (<20AA – 77AA) ACP- or SFP-Synthase

8. Available Tags and Substrate Chemistries Tags (cDNA) SNAP-tag (app 20kDa) Substrates (ex. fluors, beads) CLIP-tag (app 20kDa) Enzymes ACP-, MCP-tag (<20AA – 77AA) ACP- or SFP-Synthase

9. NEB Products using “Covalys Technology”

10. Why is it Useful? • Enables the study of “Protein Dynamics” – the study of changes in proteins, including localization, translocation, turnover, protein association, complex formation and the like. • Fundamental to academic cell biology and also of critical interest in the drug discovery arena. • Currently an emerging field, with a growing publication record and relatively few competitors. Academic/Industry sales currently at Covalys

11. Why is it Useful? “Basic” Market Dynamics • Market/trend indicator: publications on localizing proteins inside cells • Nobel prize awarded to GFP in 2008 – high visibility area Cumulative publications for GFP in “HighWire” an online scientific search engine since 2000 (50K)

12. What is the Biggest (Primary) Target Market?

13. What does a Typical “SNAP-tag” Customer Do? • Fluorescence and/or confocal microscopy, Typical filter sets • Intracellular: 488, 546, 430 nm • Extracellular: 488, 547, 647 nm Application breakdown by sales (estimated from Covalys sales). Will be complemented over time by pull-downs and in-gel assays

14. Technology Overview & Basic Workflow • Clone gene of interest into NEB expression vector • Transfect plasmid fusion into cells, protein is expressed in cells • Add label of interest • Covalent modification occurs, labeling protein • Visualize/Study 1. pSNAPm Gene of Interest 2. 4. 3.

15. Technology Overview & Basic Workflow • Clone gene of interest into NEB expression vector • Transfect plasmid fusion into cells, protein is expressed in cells • Add label of interest • Covalent modification occurs, labeling protein • Visualize/Study Live COS-7 cells expressing SNAP-Tubulin pSNAPm-Tubulin, SNAP-Cell TMR-Star

16. Applications Overview

17. Immobilization Labeling in gels Labeling in vitro Labeling in cells Purification (next year) Live Animal Imaging Multi-Functional System NEB’sProtein Tags

18. Immobilization Labeling in gels Labeling in vitro Labeling in cells Purification (next year) Live Animal Imaging Multi-Functional System – With a VERY KEY Application NEB’sProtein Tags

19. Labeling in cells Purification (next year) Live Animal Imaging Multi-Functional System – With a VERY KEY Application Labeling in gels Labeling in vitro NEB’sProtein Tags Immobilization

20. GFP (28kDa) SNAP-tag (21kDa) How do SNAP-tag and GFP Differ? More to Come… • Multifunctional tagging tool • Color from added substrate • Fluorescent upon addition of substrate • Different substrate = Different color • Fixation does not affect fluorescence • Blocking agents available • Major use is live cell imaging • Color is genetically encoded • Always fluorescent • Must re-clone to change color • Not fluorescent after fixation • No blocking agents available

21. CHO-K1 CLIP-tag CLIP-Cell TMR-Star Localization of SNAP-tag and CLIP-tag in Live Cells SNAP-tag labeling COS-7 • A useful protein tag will be localized to the appropriate sub-cellular localization of it’s fusion partner. • When SNAP-tag and CLIP-tag are expressed in cells, they are localized to the cytoplasm and the nucleus. SNAP-tag SNAP-Cell TMR-Star CLIP-tag labeling

22. Localization of SNAP-tag and CLIP-tag in Live Cells SNAP-tag labeling U-2 OS COS-7 • When SNAP-tag and CLIP-tag are fused to proteins with varied subcellular localizations, they appropriately localize (ex. surface, nucleus, mitrochondrial membrane, etc) SNAP-Tubulin, SNAP-Cell TMR-Star SNAP-ENDA-R SNAP-Surface 488 CLIP-tag labeling CHO-K1 CHO-K1 CLIP-NK1R CLIP-Surface 488 H2B-CLIP CLIP-Cell TMR-Star

23. Live Cell Staining Overview – A Simple Protocol • Dilute stock solution 1:200 in media to yield 5 uM labeling solution • Replace media on cells expressing SNAP-tag fusion, incubate 30 minutes • Wash cells 3x, incubate in fresh media for 30 minutes • Image using appropriate filter sets Cells treated with labeling media Replace with standard media, 30 minute incubation Wash 3x Incubate Image Keppler A. et al Nat Biotech 2003

24. The Products

25. NEB Products using “Covalys Technology”

26. Plasmids – Tags & Controls

27. Plasmid – Cloning Details • pSNAPm • snap26 gene, derived from human AGT • N-terminal fusion: ClaI, EcoRV (Blunt), EcoRI • C-terminal fusion: SbfI, AscI, BamHI, XhoI • pCLIPm • clip10 gene, derived from pSNAPm • Same sites as pSNAPm SNAP CLIP Gene of Interest Gene of Interest SNAP CLIP

28. Plasmid – Cloning Details • pACPm • Derived from E. coli Acyl Carrier Protein • N-terminal fusion: SacI, SacII, NotI, EcoRV, HindIII • C-terminal fusion: SbfI, AscI, BamHI • pMCPm • Derived from pACPm (D36T, D39G mutations) • Same sites as pACPm ACP MCP Gene of Interest Gene of Interest ACP MCP

29. Starter Kits – Simplify Getting Started

30. SNAP-Cell & CLIP-Cell Products • Features and Benefits • Cell permeable fluorophores • Suitable for intracellular labeling • Largest diversity of fluors available • Blocking agents available • RecommendedApplications • Live cell imaging (intracellular proteins) • Pulse chase studies • Protein localization and translocation • Fixed cell labeling

31. SNAP-, CLIP-Cell App – Intracellular Protein Trafficking Addition of substrate Cells expressing ERK2-CLIP labeled with CLIP-Cell TMR-Star (ERK2-CLIP is nuclear) Diffusion into cells PROTEIN DYNAMICS Cells expressing ERK2-CLIP labeled with CLIP-Cell TMR-Star and MEK1 (ERK2-CLIP re-localizes to the cytoplasm) Covalent attachment • Take Home Message… • Investigators can examine dynamic re-localization of proteins under a variety of stimuli Protein re-localization

32. SNAP-, CLIP-Surface Products • Features and Benefits • Non-permeable fluorophores • Suitable for cell “surface” labeling • Largest diversity of fluors available • Blocking agents available • RecommendedApplications • Live cell imaging of surface proteins • Receptor internalization • Fixed cell labeling • Biochemical assays • Flow cytometry

33. SNAP-, CLIP-, ACP-Surface App – Receptor Internalization Addition of substrate Untreated cells (membrane localized GPCR) Covalent attachment RECEPTOR INTERNALIZATION Receptor internalization Stimulated cells (internalized receptors)

34. SNAP-, CLIP-, ACP-Surface App – Receptor Internalization Addition of substrate Untreated cells expressing SNAP-ADRb2 labeled with SNAP-Surface 488 (surface localized GPCR) Covalent attachment PROTEIN DYNAMICS Receptor internalization Stimulated cells expressing SNAP-ADRb2 labeled with SNAP-Surface 488 (internalized receptors) • Take Home Message… • Investigators can examine dynamic re-localization of proteins under a variety of stimuli

35. Simultaneous Live Cell Labeling Using SNAP-and CLIP-tag SNAP-Cell 505 & CLIP-Cell TMR-Star CLIP-Cell TMR-Star SNAP-Cell 505 2. CLIP-MEK1 1. H2B-SNAP H2B-SNAP & CLIP-MEK1 • Take Home Message… • Simultaneous dual labeling of two proteins with a single system… Proteins are free to re-localize and are visualized BC BG

36. ACP-Surface Products • Features and Benefits • Non-permeable fluorophores • Very low background staining • Suitable for “surface” staining • Blocking agents available • RecommendedApplications • Live cell imaging of surface proteins • Receptor internalization • Fixed cell staining • Biochemical assays • Take Home Message… • Investigators can label sensitive membrane proteins (ex. ion channels) COS7 cells expressing ACPwt-tag-GPI anchor fusion, labeled with CoA-647, counterstained with Hoechst 33342 (blue).

37. SNAP-, CLIP-Vista & Biotin Products • Features and Benefits • Simple protocol • Suitable for use with many gel scanners • RecommendedApplications • Alternative to western blotting • Routine/High throughput analysis • Take Home Message… • Simple and rapid quantification of proteins in lysates • Features and Benefits • Flexible, compatible with streptavidin • RecommendedApplications • Multiple – cellular and biochemical • Apply proteins to streptavidin plates, etc

38. Resins and Magnetic Beads • Features and Benefits • Covalent interaction between fusion-protein and resin • Insensitive to repeated washing • RecommendedApplications • Protein pull-downs • Proteomic analysis of protein-protein interactions • Amenable to high throughput analysis • Protein purification (next year) • Take Home Message… • Pull-down assays to start… purification is coming…

39. SNAP-Cell Block + - + - MDM2 (Purified) MDM2 (Lysate) Protein Pull-downs Using SNAP-Capture Products SNAP-MDM2 + SNAP-Surface 488 SNAP-p53 + SNAP-Capture p53 p53 Sepharose Sepharose Mix Spin Wash Detect complex in SDS-Page or other fluorescent readout 488 488 MDM2 MDM2

40. HTS Binding Assays Using SNAP-tag Technologies FKBP-SNAP + SNAP-Biotin Streptavidin-coated plate Rapamycin R FKBP FKBP FRB-SNAP + SNAP-Surface 488 Biotin Biotin FKBP R Biotin 488 488 FRB FRB

41. 100 No lysate Labeling in lysate 80 60 Normalized Fluorescence 40 20 0 log (Rapamycin concentration [M]) -6 -9 -8 -7 -5 HTS Binding Assays Using SNAP-tag Technologies No interaction No fluorescence Rapamycin induced FKBP/FRB interaction FKBP Inhibition of rapamycin-mediated FKBP/FRB interaction by ascomycin Biotin Rapamycin mediated interaction Relative Fluorescence Units log (Ascomycin concentration [M]) FKBP R Biotin 488 FRB

42. Building Blocks – For Advanced Customers Only • Features and Benefits • Do-it-yourself chemistry • Couple to fluorophores or novel chemistry • Recommended to experienced users • RecommendedApplications • Same as other products – and much more • Take Home Message… • Mainly for advanced customers interested in technology development…

43. Published Applications

44. Time Time Time SNAP-tag & CLIP-tag to Follow Dynamic Processes • Single Pulse of Label: Follow protein fate over time • Blocking: Select a particular “timeslice” of protein • Pulse-Chase: “Pulse”, then “chase” in another color to investigate dynamic processes

45. Time Single pulse of label – follow protein fate over time Pulse-Labeling to Detect Protein Lifetime • Protein homeostasis: Labeling of SNAP-tag PLB and SERCA1a fusion proteins with a “pulse" of BG-Fluorescein • Results: PLB - Significant reduction in fluorescence over 24 hrs SERCA1a – Little reduction in fluorescence over 24 hrs D. L. Stenoien et al, Am. J. Physiol. Feb. 2007

46. Time Do pulse-chase labeling in different colors to investigate dynamic processes Pulse - Chase to Observe Protein Trafficking • Pulse-Chase: Labeling of SNAP-tag PLB and SERCA1a fusion proteins with a “pulse" of BG-TMR-Star and “chase” with BG-505 • Results: PLB – Newly synthesized protein localizes to perinuclear area () • SERCA1a – No evidence of cellular trafficking with time D. L. Stenoien et al, Am. J. Physiol. Feb. 2007

47. Other Applications... Limitless! • “Custom” technology development using building blocks… • Ex. localizing a novel zinc dependent fluorophore (ZP1) • Results: New application showing where zinc is localized in living cells… ZP1BG + + + + Zinc + + Zinc chelator +

48. Existing Publications… More to Come… SNAP-tag & CLIP-tag based protein labeling Gautier A. et al.:"An Engineered Protein Tag for Multiprotein Labeling in Living Cells". Chemistry & Biology 15, Feb 2008, pp. 128-136 Schulz C. and Köhn M.:"Simultaneous Protein Tagging in Two Colors", Chemistry & Biology 15, Feb 2008 Damien et al.: "Cell-surface protein-protein interaction analysis with time-resolved FRET and SNAP-tag technologies: application to GPCR oligomerization". Nature Methods. (Published online 18 May 2008), DOI: 10.1038/NMETH.1213 Mao et al.: "Optical lock-in detection of fluorescence resonance entergy transfer using synthetic and genetically encoded optical switches". Biophys J BioFAST. (June 2008), Volume 94(11), pp. 4515-24 Banala et al.: "Caged Substrates for Protein Labeling and Immobilization". Chembiochem. (Jan 2008), Volume 4;9(1), pp. 38-41 Pick H, et al.: "Distribution Plasticity of the Human Estrogen Receptor alpha in Live Cells: Distinct Imaging of Consecutively Expressed Receptors". J Mol Biol. (October 10, 2007), /Epub ahead of Print) Stenoien D. L. et al.: "Cellular Trafficking of Phospholamban and Formation of Functional Sarcoplasmic Reticulum during Myocyte Differentiation" Am J Physiol Cell Physiol (Feb 7, 2007) Jansen L. et al.: "Propagation of centromeric chromatin requires exit from mitosis" Journal of Cell Biology (March 2007); 12; 176(6): pp. 735-6. Böhme, I. et al.: "Tracking of human Y receptors in living cells – A fluorescent approach”; Peptides 28 (2007); pp. 226-234 Keppler A. et al.: "Fluorophores for live cell imaging of AGT fusion proteins across the visible spectrum" BioTechniques (Aug 2006); 41(2), pp. 167-70, 172, 174-5 Krayl M. et al.: "Fluorescence-mediated analysis of mitochondrial preprotein import in vitro" Anal Biochem (Aug 2006); 335(1), pp. 81-9 Gronemeyer T. et al.: "Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling" Prot Eng Des Sel (Jul 2006); 19(7), pp. 309-16 Tirat A. et al.: “Evaluation of two novel tag-based labeling technologies for site-specific modification of proteins” Int J Biol Macromol (Aug 2006); 39(1-3), pp. 66-76 Regoes A. et al.: "SNAP-tag mediated live cell labeling as an alternative to GFP in anaerobic organisms" BioTechniques (Dec 2005); 39(6), pp. 809-12 Keppler A. et al.: "Labeling of fusion proteins with synthetic fluorophores in live cells" PNAS (2004); 101, pp. 9955-9 Kindermann M. et al.: "Synthesis and characterization of bifunctional probes for the specific labeling of fusion proteins" Bioorg Med Chem Lett (2004); 14, pp. 2725-8 Keppler A. et al.: "Labeling of fusion proteins of O(6)-alkylguanine-DNA alkyltransferase with small molecules in vitro and in vivo" Methods (2004); 32, pp. 437-44 Juillerat A. et al.: "Directed evolution of O(6)-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo" Chem Biol (2003); 10, pp. 313-7 Keppler A. et al.: "A general method for the covalent labeling of fusion proteins with small molecules in vivo" Nature Biotechnology (2003); 21, pp. 86-9 SNAP-tag used for protein immobilization Gronemeyer Th. et al.: „A new pull-down assay to monitor protein-protein interactions” HUPO-2007 poster contribution Jongsma M. A. and Litjens R. H.: "Self-assembling protein arrays on DNA chips by auto-labeling fusion proteins with a single DNA address" Proteomics (May 2006); 6(9), pp. 2650-5 Sielaff I. et al.: "Protein function microarrays based on self-immobilizing and self-labeling fusion proteins" ChemBioChem (Jan 2006); 7(1), pp. 194-202 Kufer S. K. et al.: "Covalent immobilization of recombinant fusion proteins with hAGT for single molecule force spectroscopy" Eur Biophys J (2005); 35(1), pp. 72-8 Tugulu S. et al.: "Protein-Functionalized Polymer Brushes" Biomacromolecules (2005); 6(3), pp. 1602-7 Huber W. et al.: "SPR-based interaction studies with small molecular weight ligands using hAGT fusion proteins" Anal Biochem (2004); 333(2), pp. 280-8 Kindermann M. et al.: "Covalent and selective immobilization of fusion proteins" JACS (2003); 125(26), pp. 7810-1 SNAP-tag - other applications Lemercier, G. et al.: "Inducing and Sensing Protein–Protein Interactions in Living Cells by Selective Cross-linking" Angew Chem Int Ed (2007); 46, in press (DOI:10.1002/anie.200700408) Gendreizig, S. et al.: "Induced protein dimerization in vivo through covalent labeling" JACS (2003); 125(49), pp. 14970-1 Reviews and background papers on Covalys’ technology Johnsson N. et al.: "Protein chemistry on the surface of living cells" ChemBioChem (2005); 6(1), pp. 47- 52 (Review) Gronemeyer T. et al: "Adding value to fusion proteins through covalent labeling" Curr Opin Biotechnol (Aug 2005); 16(4), pp. 453-8 Michnick, S. W.: " Research Focus: Proteomics in Living Cells” Drug Discovery Today (Mar 2004); 9(6), pp. 262-7 Johnsson N. and Johnsson K.: "A fusion of disciplines: chemical approaches to exploit fusion proteins for functional genomics" ChemBioChem (Sep 5, 2003); 4(9), pp. 803-10 ACP-tag Zhou Z. et al.: "Genetically Encoded Short Peptide Tags for Orthogonal Protein Labeling by Sfp and AcpS Phosphopantetheinyl Transferases" ACS Chemical Biology Vol. 2 (5),(2007) pp. 337-346 Jacquier V. et al.: "Visualizing receptor trafficking in living cells down to the single-molecule level" PNAS (Sep 2006); 103(39), pp. 14325-30 Prummer M. et al.: "Post-translational covalent labeling reveals heterogeneous mobility of individual G protein-coupled receptors in living cells" ChemBioChem (2006); 7, pp. 908-11 Meyer B. H. et al.: "Covalent labeling of cell-surface proteins for in-vivo FRET studies" FEBS Letters (Mar 2006); 580(6), pp. 1654-8 Meyer B. H. et al.: "FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells" PNAS (Feb 14, 2006); 103(7), pp. 2138-43 Sielaff I. et al.: "Protein function microarrays based on self-immobilizing and self-labeling fusion proteins" ChemBioChem (Jan 2006); 7(1), pp. 194-202 Vivero-Pol L. et al.: "Multicolor imaging of cell surface proteins" JACS (2005); 127(37), pp. 12770-1 Yin J. et al.: "Single-Cell FRET Imaging of Transferrin Receptor Trafficking Dynamics by Sfp-Catalyzed, Site-Specific Protein Labeling" Chem Biol (2005); 12(9), pp. 999-1006 Cravatt B. F. Comment: "Live Chem. Repts from Cell Surface" Chem Biol (2005); 12(9), pp. 954-956 George N. et al.: "Spec. lblg of cell surf. Prot. w. chem. diverse cmpds" JACS (2004); 126, pp. 8896-7 Yin J. et al.: "Lblng prot. w. small molec. by site-spec. posttransl. mod." JACS (2004); 126, pp. 7754-5 La Clair J. J. et al.: "Manip. of carrier prot. in antibiotic biosynth" Chem Biol (2004); 11(2), pp. 195-201 • vs. Competition • vs. GFP… much LESS – more to do! • vs. Halo-tag, LigandLink, Lumio = More!

49. Competitive Landscape & Marketing Approach

50. Competitive Landscape • GFP – Dominant in publications and free for academics. • Antibodies – Great in fixed cells but can’t be used in lived cells. • Other chemical labeling approaches? Yes, but few publications…