A Fusion Tag for Complete Protein Characterization. Rob Chumanov, PhD, MBA

Transcription

1 A Fusion Tag for Complete Protein Characterization Rob Chumanov, PhD, MBA 1

2 Outline A Single Fusion Tag for All Your Protein Analysis 1. What is HaloTag technology? 2. Why use HaloTag for protein characterization? 3. Function of proteins in isolation Protein purification from mammalian cells 4. Function of proteins within cells Live cell Imaging Capturing Protein-Protein Interactions (PPI s) Live cell PPI assays with NanoBRET 5. Getting started with HaloTag technology HaloTag 2

3 HaloTag Technology A Single, Multifunctional Protein Fusion Tag Proteomic Analysis Protein interactions Purified protein activity PTMs Proteins in isolation Cell-Based Analysis Localization Real-time imaging Protein trafficking Live-cell assays Proteins in cells Affinity tags (e.g. GST, Flag, His) Fluorescent proteins (e.g. GFP, RFP) Antibodies HaloTag 3

4 Covalent Binding Mechanism of HaloTag Technology HaloTag is a protein fusion tag engineered to covalently bind a synthetic chloroalkane ligand. Chloroalkane Functional group Protein of Interest HT + Cl - Surface attachment & Fluorescent ligands Protein of Interest HT Covalent bond = Irreversible attachment!!! 4

5 Multiple HaloTag Ligands for Every Application HaloTag HaloTag ligand HaloTag fluorescent ligands Multiple fluorophores Cell-permeable ligands Cell-impermeable ligands Red-shifted ligands HaloTag surfaces Nonmagnetic resin Magnetic resin Glass slides 96-well plates R HaloTag reactive ligands Reactive chemistry (i.e. amine, thiol) Attach to functional groups of choice (i.e. Quantum dots, PET ligands) 5

6 Make and Use One Clone for Multiple Applications! HaloTag Vector POI ORF Expression of HaloTag fusion HaloTag ORF Covalent Capture HaloTag Surfaces Protein Labeling HaloTag Fluorescent Ligands O O O O Protein Display Interactions Purification Detection Cellular Imaging NanoBRET 6

7 Simple and Gentle Protein Purification Protocol Lyse HT + + Bind Wash Transfect Cl HaloLink Cl Cl Cl + HT Cleave/Capture TEV Lysis/binding buffer: PBS Pure POI 7

8 Minimal Non-specific Binding with HaloTag Resin Cytoplasmic Lysate + Resin 1 hr Sample buffer SDS-PAGE Coomassie or Silver Stain Magnetic Non-Magnetic 8

9 Optimization of Transient Protein Expression Using Fluorescent HaloTag Ligands Transfection in 24-well plates 100 nm TMRDirect in media POI HT O O PKCg PKAc Src DEGFR PI3kg Analysis of expression level cell to gel analysis Hours Convenient fluorescent detection of expressed HaloTag fusion proteins Rapid optimization of expression conditions 9

10 Highly Efficient Protein Capture and Recovery of Human Kinases from HEK293T Cells PKCg PKAc Src DEGFR PI3Kg T FT Y T FT Y T FT Y T FT Y T FT Y T: starting material FT: unbound Y: yield 2x10 8 cells Kinases PKCg PKAc SRC DEGFR PI3Kg Estimated POI expression (mg) Yield (mg) % Recovery 84% 88% 76% 94% 89% 10

11 HaloTag Enables Greater Recovery and Purity of Purified Proteins PRKCg PI3Kg kda Protein Recovery (%) Tag PKCg PI3Kg HaloTag FLAG xFLAG His 6 Tag

12 HaloTag Core Advantages for Purification Simple protocol that uses gentle purification conditions (PBS buffer) High recovery and purity even with poorly expressed proteins Significantly more economical than antibody-based tags (HA, myc, Flag) Functions as a solubility and purification tag in E. coli expression systems 12

13 Isolated Proteins are Not Physiologically Relevant Replication Transcription Translation Cell growth Signal transduction etc Need powerful in vitro and live cell tools to characterize protein function 13

14 Outline A Single Fusion Tag for All Your Protein Analysis 1. What is HaloTag technology? 2. Why use HaloTag for protein characterization? 3. Function of proteins in isolation Protein purification from mammalian cells 4. Function of proteins within cells Live cell Imaging Capturing Protein-Protein Interactions (PPI s) Live cell PPI assays with NanoBRET 5. Getting started with HaloTag technology HaloTag 14

15 Make and Use One Clone for Multiple Applications! HaloTag Vector POI ORF Expression of HaloTag fusion HaloTag ORF Covalent Capture HaloTag Surfaces Protein Labeling HaloTag Fluorescent Ligands O O O O Protein Display Interactions Purification Detection Cellular Imaging NanoBRET 15

16 Fluorescent Labeling and Detection Generate Multiple Colors 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 16

17 Easily Track Protein Translocation in Real Time Signal (TNFa) (0 min) p50 p65 IkB (80 min) (5 min) p50 p65 p50 p65 (10 min) IkB 0 min 5 min 10 min 20 min 40 min 60 min 80 min 100 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 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):

18 Real Time Imaging of HaloTag β1 Integrin Fragment Label with nonpermeant Green Label with permeant HaloTag TM TMR Wash Image Incubate 12 hrs Image Vector Time points (3-9) collected for gel Transfect cells 12hrs Time Surface Svendsen, S., et al. BMC Cell Biol, 9: 17, impermeable - permeable Spatial control of labeling Follow protein trafficking 18

19 Trafficking of GABA A Receptor, a Ligand-gated Ion Channel Super Family a g - non-permeable - permeable γ2l-halotag co-expressed with α1 or b2 does not traffic to cell surface γ2l-halotag co-expressed with both α1 and β2 traffics to cell surface GABA A R pharmacology is not affected In collaboration with Srinivasan Venkatachalan and Cynthia Czajkowski 19

20 HaloTag is Fully Compatible with Other Live-cell and Fixed Cell Imaging Techniques HaloTag NLS 3 -TMR ligand hmgfp α-tubulin p65-halotag-tmr Ligand Alexa 488-β-tubulin Ab U2OS cells with HaloTag NLS 3 labeled with R110Direct Live Cells Fixed Cells Flow Cytometry HaloTag is compatible with fluorescent protein fusions HaloTag labeling is compatible with fixation, ICC (can use both a ligand and an antibody), and flow cytometry and FACS Change HaloTag color based on experimental need 20

21 HaloTag Core Advantages for Imaging Labeling only images protein at that point in time Protein expressed after initial labeling is not visualized (unless labeled again) Enables post-chase labeling experiments (spatial and temporal trafficking) Label extracellular and intracellular HaloTag fusion proteins in a single experiment with different fluorophores Label remains bright in both live and fixed cells and is compatible with traditional imaging techniques Ability to change the fluorophores adds greater experimental flexibility HaloTag fusion proteins are compatible with other fluorescent proteins 21

22 Make and Use One Clone for Multiple Applications! HaloTag Vector POI ORF Expression of HaloTag fusion HaloTag ORF Covalent Capture HaloTag Surfaces Protein Labeling HaloTag Fluorescent Ligands O O O O Protein Display Interactions Purification Detection Cellular Imaging NanoBRET 22

23 Correlate Imaging Results to Biochemistry Data min Binding to IkB Protein Imaging HT p min Anti IkB immunoblot Binding to IkB Promoter min PCR 23

24 Minimal Non-specific Binding with HaloTag Resin Cytoplasmic Lysate + Resin 1 hr Sample buffer SDS-PAGE Coomassie or Silver Stain Magnetic Non-Magnetic 24

25 Capture Protein Interactions with a Simple Protocol and Controls HaloTag fusion construct HT POI Express HaloTag control construct HT Halo Link HT Lyse/Bind to Resin/Wash POI Halo Link HT TEV Cleavage SDS Elution Release/Elute TEV Cleavage SDS Elution POI Avoid swamping MS with Bait Negative control 25

26 Eukaryotic RNA Polymerase Complexes with Multiple Unique and Shared Components Large complexes, with multiple subunits Several common proteins HaloTag on N-terminus of POLR2H (based on structure) Imaging validation for correct localization RNAP II POL2RH-Halo 26

27 Improved Pull-down of Eukaryotic RNA Polymerase Complex with HaloTag 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 PolR1E X X hrpa34 X X X X ZNRD1 X X X TWISTNB X X PolR2A X X X X PolR2B X X X X PolR2C X X X X PolR2D X X X PolR2E X X X 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 PolR3C X X X X PolR3D X X X X PolR3E X X X PolR3F X X PolR3G X X X PolR3H X X X PolR3K X X X PolR3I X X X POLR2H- HT Fusion Endogenous POLR2H p21 p22 p23 p24 High Mid Low Ultra POLR2H- FLAG Fusion Endogenous POLR2H Western Blots Against PolR2H

28 How Can We See Protein:Protein Interactions? Bioluminescence Resonance Energy Transfer (BRET) HaloTag fusion construct HT POI NanoLuc (Nluc) HT Halo Link HT POI NanoLuc Luciferase Ultra small luciferase (19kDa) Greater than 100X brighter than Rluc and Fluc 28

29 NanoLuc HaloTag BRET for Live-cell Protein:Protein Interaction Studies NanoBRET 618nm HaloTag Ligand Protein A HT + Protein A HT Cl - HaloTag Technology Advantage: Selection of perfect resonance energy acceptor NanoBRET 618nm ligand Acceptor Validation through imaging and biochemical pull-downs Protein B NLuc Protein B NLuc BRET Protein A HaloTag Protein A HaloTag 29

30 NanoBRET Assay Schematic and No-Wash Protocol 1. Bulk transfection FuGENE HD HaloTag fusion H3 or H4 NanoLuc fusion NanoLuc HT BRET Ligand NanoLuc Substrate BRD4 2. Re-plating +/- low concentration BRET ligand in phenol-red free media 3. Optional compound treatment 4. Furimazine addition and measurement Measure emissions and calculate ratio Instrument capable of reading ~450nm and ~610nm emissions 30

31 S /B R a tio NanoBRET is Substantially More Efficient than Traditional BRET1 Systems F r b -N L u c / F K B P -H T F r b -R L u c 8 / F K B P -Y F P R a p a m y c in [n M ] 31

32 BRET Ratio NanoBRET for Glucocorticoid Receptor (GR) Dimerization GR GR HT NLuc NanoBRET 618nm ligand + Dexa - methasone GR GR HT NLuc BRET Translocation into nucleus cytosol nucleus nm [Dexamethasone] 32

33 Corrected BRET Ratio Corrected BRET Ratio Corrected BRET Ratio Demonstrating Compound Specificity with NanoBRET Assays JQ1 potency differences amongst bromodomain family members (BRD4 and CBP) and histones NLuc-BRD4 BRD4 : Histone H3.3 BRD4 : Histone H4 CBP : Histone H4 IC50 = 52 nm IC50 = 228 nm 33

34 Only Full-length Proteins in Live Cells Present a Physiologically Accurate Picture MBD PHD BRD BAZ2A Full-length MBD+PHD+BRD PHD+BRD BRD Addition of PHD domain significantly reduces inhibition by GSK

35 HaloTag Provides a Powerful Tool to Capture/Characterize the Protein Interactome Low non-specific binding Covalent capture-no leaching Specific elution-no bait release and Live Cell Protein:Protein Interaction Assays with NanoBRET 35

36 Outline A Single Fusion Tag for All Your Protein Analysis 1. What is HaloTag technology? 2. Why use HaloTag for protein characterization? 3. Function of proteins in isolation Protein purification from mammalian cells 4. Function of proteins within cells Live cell Imaging Capturing Protein-Protein Interactions (PPI s) Live cell PPI assays with NanoBRET 5. Getting started with HaloTag technology HaloTag 36

37 Getting Started With HaloTag Techology Many Options are Available Cost $$ Effort Purchase/Fast Pre-made full-length human ORF clones Flexi vector cloning system FREE Clone/Slower HaloTag vector card with traditional (MCS) vectors 37

38 Find My Gene on An Easy to Use Gene/Protein Information Tool Find My Gene Find My Gene 38

39 Expression-validated Human ORF Clones Full length human ORF clones, N- terminally tagged with HaloTag CMV-driven mammalian overexpression constructs ORF expression validated in HEK 293 cells: in-gel analysis and imaging Gene Family Available Genes All Genes 9,100 Transcription factors 906 Kinase 786 Ubiquitin-related 741 GPCRs 632 Epigenetics-related 580 RNA Binding 476 Transporters 412 Cytokines 272 Proteases 270 Growth Factors 270 Ion Channels 147 Phosphatases 88 Nuclear Receptors 45 39

40 Flexi -vector Cloning System Gets You Started with HaloTag N-terminal transfer Directional cloning based method for protein-coding DNA sequences Uses rare-cutting restriction enzymes C-terminal transfer Rapidly move protein-coding regions without resequencing Flexi vectors for HaloTag, HIS, GST tag expression systems Vectors for mammalian, E. coli, cell-free expression 40

41 Receive FREE HaloTag Expression Vectors by Mail Card sent by post mail that contains imbedded plasmid DNA Three expression vectors: N- and C- terminal fusion vectors for mammalian expression N-terminal HaloTag for E.coli expression HaloTag vector card Learn more at 41

42 Experience the Power of a Single Tag From work on isolated proteins to Live-cell HTS protein:protein interactions assays and everything in between HaloTag 42

43 Have Questions? Technical Service Scientists Are Ready to Help! By phone: Available 7am-6pm Central M-F Online Available 7am-6pm Central M-F Global Chat with Branch office tech serv scientists, too, after hours (language dependent) Guaranteed answers within 24hr Most responses within 2hrs 43