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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
• Protein:DNA interactions
• Protein Purification
• Localization
• Real-time imaging
• Protein trafficking
• Fluorescent Imaging
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
Protein of
Interest
Functional group
+
HT
Surface attachment
&
Fluorescent ligands
ClProtein 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
 Cell-permeable ligands
 Cell-impermeable ligands
HaloTag
HaloTag surfaces
 Nonmagnetic resin
 Magnetic resin
 Glass slides
R
©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.
HaloTag reactive ligands
 Reactive chemistry (i.e. amine , thiol)
 Attach to functional groups of choice
(i.e. Quantum dots, PET ligands)
One Genetic Construct for Multiple Applications
Do It All with One Clone!
HaloTag
POI
Vector
HaloTag
Expression of HaloTag fusion
Covalent capture
Protein labeling
HaloTag surfaces
HaloTag fluorescent ligands
O
O
OO
Protein display
Interactions
©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.
Purification
Detection
Cellular imaging
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
Lysate
+
Resin
1 hr
Sample buffer
SDS-PAGE
Coomassie
©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.
Silver
Silver Staining
Protein:Protein Interaction Capture
Simple Protocol and Controls
HaloTag
fusion
construct
POI
Express
HT
Halo
Link
HaloTag
control
construct
HT
Lyse/Wash
HT
Halo
Link
POI
TEV
Cleavage
SDS
Elution
POI
©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.
Release/Elute
TEV
Cleavage
HT
SDS
Elution
Negative control
Protein:Protein Interaction Capture
SDS Elution Only Releases Interacting Proteins
HaloTag
fusion
construct
M
Stable HEK293
p65 CTL
POI
HT
220
120
100
80
Halo
Link
60
HT
50
40
POI
30
TEV
Cleavage
20
SDS
Elution
POI
©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.
Avoid swamping
MS with Bait
MS/MS Identification:
P105, P100, Rel A, Rel B, C-Rel
IκBα, IκBβ, and IκBε
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
Wash
Image
©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.
HaloTag - NLS
Mitochondria
Cytosol
Membrane
Mito - HaloTag
p65 -HaloTag
HaloTag - ECS
Real Time Imaging of HaloTag® - NF-κB Construct
Easily Track Translocation
Signal (TNFa)
(0 min)
p50
(5 min)
p50
p65
p65
IkB
IkB
(10 min)
(80 min)
0 min
60 min
5 min
80 min
p50
p65
10 min
100 min
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.
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
translocation kinetics
Imaging and Interaction Results Correlate
min
Binding to IkB protein.
0
Protein:protein
10
Anti IkB immunoblot
0
30
60
90 min
Imaging
HT
p65
30
Binding to IkB promoter.
PCR
Protein:DNA
0
15
30
45
60 min
©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.
110
Translocation
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
PolR1A
X
X
PolR1B
X
X
PolR1C=RPAC1
X
X
PolR1D=RPAC2
X
X
PolR1E
X
hRPA34
X
X
ZNRD1
X
X
TWISTNB
X
X
PolR2A
X
X
PolR2B
X
X
PolR2C
X
X
PolR2D
X
X
PolR2E
X
X
PolR2F
X
PolR2G
X
X
PolR2H
X
X
PolR2I
X
X
PolR2J
X
X
PolR2K
X
X
PolR2L
X
X
PolR3A
X
X
PolR3B
X
X
PolR3C
X
X
PolR3D
X
X
PolR3E
X
X
PolR3F
X
X
PolR3G
X
X
PolR3H
X
X
PolR3K
X
X
©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.
PolR3I
X
X
HT Ultra
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
FLAG
X
X
X
p21 p22 p23 p24
220
High Med
120
100
80
Low Ultra
X
X
X
X
POLR2H- HT
Fusion
60
50
40
30
Endogenous
POLR2H
20
X
X
X
X
POLR2H- FLAG Fusion
Endogenous POLR2H
Western blots against PolR2H
Eukaryotic RNA Polymerase Complex
Associated Proteins
Associated Factor HT High
CDC73
X
EFXAB1/TUD2
X
ELAVL-1
X
Elongin B
X
Elongin C
MED25
MGC14560
X
Monad
X
NOP17
X
PDRG1
X
RPAP2
X
RPAP3
X
RUVBL1
X
RUVBL2
X
TAF(II)210
X
TAF6L
Tat-SF1
X
18
15
©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.
HT Mid HT Ultra FLAG
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
p21 p22 p23 p24
220
High Med
120
100
80
Low Ultra
POLR2H- HT
Fusion
40
X
30
Endogenous
POLR2H
X
X
X
14
X
8
60
50
5
Daniels, D. et al J. Proteome Res. 2012, 11, 564-575.
20
The Ribosome Story
One of the Most Important Macromolecular Machines
One of the most important
macromolecular machines
in the cell.
Prokaryotic 70S Ribosome
Modeled with HaloTag
Challenges in the field:




Difficult to study interactions
and intermediates.
Sucrose gradients not discrete.
Protein localization, trafficking,
and turnover important –
difficult with GFP.
Single molecule community
needs better handle for surface
attachment.
Placed HaloTag on 40S
subunit protein, Human
RPS9.
©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.
TEV
HaloTag
Selmer et al. (2006) Science 313, 1935 pdb:2j00 and 2j01 – 70S Ribosome
Monitoring Ribosomal Trafficking and Populations
HaloTag® Enables Complex Trafficking Experiments
Serum
Starve
24hrs
TMR
Red
Label
Old population
24hr
Wash
New population
Add back
Serum
3hrs
Oregon
Green
Label
Old and new
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
RNA Capture
HT Ctrl
60S
HaloLink
Resin
40S
220
120
100
HT
TEV Cleavage
80
60
?
60S
40S
50
S9
40
40S
30
60S
40S
20
Identified by MS (LC-MS/MS)
•31 of 33 40S proteins
•42 of 50 60S proteins
•2 Poly-A binding proteins
•1 GNF exchange protein
•9 Nuclear ribonucleoproteins
•2 Initiation factors
•2 Elongation factors
•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.
Purified
RNAs
28S
18S
5.8S
5S
Elongation of Full-length Protein – Luciferase
Captured Complexes are Translational Active
Captured
S9-HT Ribosome with luciferase mRNA
(HEK293 Luciferase Stable)
Detection of Luciferase
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 KRas 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®
•
•
•
•
©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.
Expressed H1.0 HaloTag in E. coli
Immobilize on HaloLink resin
Incubate with nuclear lysates
Capture interacting nuclear proteins
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 Gene™ 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
•
©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.
Align available ORF clones with inputed
sequences
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
In-gel analysis with HaloTag® ligands
©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.
Imaging validation
Using the HaloTag® ligands
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
All Genes
Transcription factors
Kinase
Ubiquitin-related
GPCRs
Epigenetics-related
RNA Binding
Transporters
Cytokines
Proteases
Growth Factors
Ion Channels
Phosphatases
Nuclear Receptors
©2013 Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.
Available Genes % of total Total Human ORFs
9,100
906
786
741
632
580
476
412
272
270
270
147
88
45
42%
84%
84%
83%
64%
78%
57%
38%
82%
48%
91%
40%
55%
92%
21,658
1076
936
888
993
743
839
1079
331
565
296
364
160
49
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.