How to Identify Physiologically Relevant 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 (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 Lysate + Resin

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 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 construct • SW-48 cell line with native K- Ras promoter • Endogenous expression levels

Nature, 2009 ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Endogenous Level Expression of K-Ras in SW48 Cells

Co-staining with DNA stain

Co-staining with membrane stain

©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Validation of K-Ras Interacting Proteins with HaloTag® – K-Ras Pull-down

• Endogenous expression • Other interactions – publication in progress

©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Visualizing the H1.0 Interactome with HaloTag®

• Expressed H1.0 HaloTag in E. coli • Immobilize on HaloLink resin • Incubate with nuclear lysates • Capture interacting nuclear proteins

©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Histone H1 Interactome Identified Known and Novel Interactors

• Validated known interactions • Captured novel interactions

©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Find My ™ on www.promega.com An Easy to Use Gene/Protein Information Tool

Find My Gene™

Find My Gene™

©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Getting Started with Pre-made ORF Clones Identify Them at Find My Gene

www.promega.com • Allows for variety of search criteria • BLAST, key word, synonym • Links to protein information • UniProt, NCBI • Information on interaction networks • NCBI Interactome • Align available ORF clones with inputed sequences

©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. ORF Clones

• Full – length human ORF clones • ORFs are N-terminally tagged with HaloTag® • CMV-driven mammalian overexpression constructs • ORF expression validated in HEK 293 cells

Expression validation Imaging validation In-gel analysis with HaloTag® ligands Using the HaloTag® ligands

©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. The ORF Collection Over >9,100 HaloTag®-ORF Clones Available

• Unique gene-centric scientific destination on www.promega.com • All expression-validation data is available through Find My Gene™

Gene Family Available % of total Total Human ORFs All Genes 9,100 42% 21,658 Transcription factors 906 84% 1076 Kinase 786 84% 936 Ubiquitin-related 741 83% 888 GPCRs 632 64% 993 Epigenetics-related 580 78% 743 RNA Binding 476 57% 839 Transporters 412 38% 1079 Cytokines 272 82% 331 Proteases 270 48% 565 Growth Factors 270 91% 296 Ion Channels 147 40% 364 Phosphatases 88 55% 160 Nuclear Receptors 45 92% 49 ©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Why Kazusa Clones and Find My Gene™

• Mammalian expression validated clones

• All validation data is available for inspection: sequencing and expression

• Visual guarantee that the purchased HaloTag clone will work with HaloTag reagents

©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure. Summary - Why HaloTag®? Capture What You’ve Been Missing

• Low non-specific binding • Covalent capture-no leaching • Specific elution-no bait release

www.biomedcentral.com Capture what you have been missing with traditional technologies

©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.