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How to Identify Physiologically Relevant Protein Interactions Using Covalent-Capture Halotag® Technology Rob Chumanov, Phd, MBA

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How to Identify Physiologically Relevant Protein Interactions Using Covalent-Capture Halotag® Technology Rob Chumanov, Phd, MBA How to Identify Physiologically Relevant Protein Interactions Using Covalent-capture HaloTag® Technology Rob Chumanov, PhD, MBA ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Outline 1. What is HaloTag® technology? 2. Why use HaloTag for pull-down assays? 3. Capturing small and large protein complexes 4. Studying physiologically relevant protein:protein interactions 5. FindMyGene™ Promega-Madison ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. HaloTag® Platform One Fusion Protein Tag for Multiple Apps Proteomic Analysis Cell-Based Analysis In-vivo models • Protein:Protein interactions • Localization • Fluorescent Imaging • Protein:DNA interactions • Real-time imaging • Protein Purification • Protein trafficking Fluorescent proteins (e.g. GFP, RFP) Affinity tags (e.g. GST, Flag, His) Antibodies HaloTag® ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. HaloTag™ Technology Covalent Binding Mechanism • HaloTag is a protein fusion tag engineered to covalently bind a synthetic chloroalkane ligand. Chloroalkane Functional group Protein of HT Interest + Surface attachment & Cl- Fluorescent ligands Protein of Interest HT Covalent bond = Irreversible attachment!!! ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. HaloTag™ Ligand Diversity Multiple Ligands to Meet Your Application Needs HaloTag ligand HaloTag fluorescent ligands . Multiple fluorophores HaloTag . Cell-permeable ligands . Cell-impermeable ligands HaloTag surfaces . Nonmagnetic resin . Magnetic resin . Glass slides HaloTag reactive ligands . Reactive chemistry (i.e. amine , thiol) R . Attach to functional groups of choice (i.e. Quantum dots, PET ligands) ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. One Genetic Construct for Multiple Applications Do It All with One Clone! HaloTag Vector POI HaloTag Expression of HaloTag fusion Covalent capture Protein labeling HaloTag surfaces HaloTag fluorescent ligands O O O O Protein display Interactions Purification Detection Cellular imaging ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Proteins Rarely Work in Isolation Replication Transcription Translation Cell growth Signal Transduction etc… www.biomedcentral.com Need powerful tools to capture and characterize the protein interactome ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. HaloTag® Provides a Powerful Tool to Capture/Characterize the Protein Interactome • Low non-specific binding • Covalent capture-no leaching • Specific elution-no bait release www.biomedcentral.com ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Magnetic Resin Comparison Minimal Background Binding with HaloTag® Resin Cytoplasmic + Resin Lysate 1 hr Sample buffer SDS-PAGE Coomassie Silver Coomassie Staining ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Non-Magnetic Resin Comparison Minimal Background Binding with HaloTag® Resin Cytoplasmic + Resin Lysate 1 hr Sample buffer SDS-PAGE Coomassie Silver Silver Staining ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Protein:Protein Interaction Capture Simple Protocol and Controls HaloTag HaloTag fusion control construct POI construct HT Express HT Halo Lyse/Wash Link HT Halo POI Link HT TEV TEV SDS SDS Release/Elute Cleavage Cleavage Elution Elution POI Negative control ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Protein:Protein Interaction Capture SDS Elution Only Releases Interacting Proteins Stable HEK293 HaloTag M p65 CTL fusion construct POI HT 220 120 100 80 60 Halo Link HT 50 40 POI 30 20 TEV SDS Cleavage Elution Avoid swamping POI MS with Bait MS/MS Identification: P105, P100, Rel A, Rel B, C-Rel IκBα, IκBβ, and IκBε ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Fluorescent Labeling and Detection Easy Imaging with the Same Fusion Tag Transfect / Express Labeling with multiple fluorophores HaloTag fusion to nuclear localization sequence (NLS) -HEK293T cells Label Localization to different cellular compartments Nucleus Mitochondria Cytosol Membrane Wash Image HaloTag - NLS Mito - HaloTag p65 -HaloTag HaloTag - ECS ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Real Time Imaging of HaloTag® - NF-κB Construct Easily Track Translocation Signal (TNFa) (0 min) (5 min) p50 p50 p65 IkB p65 IkB (10 min) (80 min) p50 p65 0 min 5 min 10 min 20 min 40 min • HeLa cells expressing p65-HaloTag labeled with TMR Ligand • Treated with TNFα (at 0 min) and imaged every 5 min for 120 min • HaloTag has no effect on protein 60 min 80 min 100 min translocation kinetics GV Los, et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol, Jun 2008; 3(6): 373-82. ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Imaging and Interaction Results Correlate min Translocation Binding to IkB protein. 0 Protein:protein Anti IkB immunoblot 10 0 30 60 90 min HT Imaging p65 Binding to IkB promoter. 30 PCR Protein:DNA 110 0 15 30 45 60 min ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Eukaryotic RNA Polymerase Complexes Multiple Unique and Shared Components • Large complexes, with multiple subunits • Several common proteins • HaloTag on N-terminus of POLR2H (based on structure) ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. POLR2H-HaloTag Properly Localizes to Nucleus RNAP II POL2RH-Halo Overlay • POLR2H-HaloTag is properly localized to the nucleus. • Co-localization with antibody specific to the CTD of POLR2A. ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Eukaryotic RNA Polymerase Complex Subunit HT High HT Mid HT Ultra FLAG PolR1A X X X X PolR1B X X X X PolR1C=RPAC1 X X X X PolR1D=RPAC2 X X X p21 p22 p23 p24 220 PolR1E X X High Med Low Ultra 120 100 hRPA34 X X X X 80 ZNRD1 X X X POLR2H- HT 60 TWISTNB X X 50 PolR2A X X X X Fusion 40 PolR2B X X X X PolR2C X X X X 30 PolR2D X X X Endogenous 20 PolR2E X X X POLR2H PolR2F X PolR2G X X X PolR2H X X X X PolR2I X X X PolR2J X X X PolR2K X X X PolR2L X X X PolR3A X X X X PolR3B X X X POLR2H- FLAG Fusion PolR3C X X X X Endogenous POLR2H PolR3D X X X X PolR3E X X X Western blots against PolR2H PolR3F X X PolR3G X X X PolR3H X X X PolR3K X X X ©2013PolR3I Promega Corporation. Confidential and Proprietary.X Not for Further Disclosure.X X Eukaryotic RNA Polymerase Complex Associated Proteins Associated Factor HT High HT Mid HT Ultra FLAG CDC73 X X X EFXAB1/TUD2 X X ELAVL-1 X Elongin B X X Elongin C X p21 p22 p23 p24 220 MED25 X High Med Low Ultra 120 100 MGC14560 X X X 80 Monad X X X POLR2H- HT 60 50 NOP17 X X Fusion 40 PDRG1 X X X 30 RPAP2 X X X X Endogenous RPAP3 X X POLR2H 20 RUVBL1 X X X X RUVBL2 X X X X TAF(II)210 X TAF6L X Tat-SF1 X X 18 15 14 8 5 ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Daniels, D. et al J. Proteome Res. 2012, 11, 564-575. The Ribosome Story One of the Most Important Macromolecular Machines One of the most important macromolecular machines Prokaryotic 70S Ribosome Modeled with HaloTag in the cell. Challenges in the field: . Difficult to study interactions and intermediates. Sucrose gradients not discrete. TEV . Protein localization, trafficking, and turnover important – difficult with GFP. Single molecule community needs better handle for surface HaloTag attachment. Placed HaloTag on 40S subunit protein, Human RPS9. Selmer et al. (2006) Science 313, 1935 pdb:2j00 and 2j01 – 70S Ribosome ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Monitoring Ribosomal Trafficking and Populations HaloTag® Enables Complex Trafficking Experiments Serum TMR Add back Oregon Starve Red Wash Serum Green 24hrs Label 3hrs Label Old population New population Old and new 24hr 3hr H T • Newly synthesized ribosomal proteins localized to nucleoli 3hrs post-stress 24hr • After 24hrs of recovery, ribosomal populations re-localized to cytoplasm ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. HaloTag® Pull-down Using Ribosomal RPS9 Efficient Capture of a Huge Protein Complex S9-HT Pull-down TEV Cleavage S9-HT HT Ctrl RNA Capture 60S Purified HaloLink 40S RNAs Resin 220 Identified by MS (LC-MS/MS) 120 HT TEV Cleavage 100 •31 of 33 40S proteins 28S 80 •42 of 50 60S proteins 60 • 2 Poly-A binding proteins 18S 50 •1 GNF exchange protein ? 5.8S S9 40 •9 Nuclear ribonucleoproteins 60S 5S 60S •2 Initiation factors 40S 40S 30 40S •2 Elongation factors 20 •2 Splicing factors Silver Stain • Purified the 80S ribosome directly from the HeLa cell lysate. • Identified 19 interacting partners. ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Elongation of Full-length Protein – Luciferase Captured Complexes are Translational Active Captured S9-HT Ribosome with luciferase mRNA Detection of Luciferase (HEK293 Luciferase Stable) HaloLink Resin HT TEV Cleavage Add tRNAs Luciferase • Isolated ribosomes show capture of Luc-mRNA and complete elongation of luciferase ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Sensitive Capture with of K-Ras Interacting Partners at Endogenous Expression Levels • K-Ras – HaloTag
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