accelerating proteomics research with a unique, multi

©2012 Promega Corporation

Accelerating Proteomics Research with a Unique, Multi-functional Fusion Protein Technology and an Extensive Collection of Premade Clones

Jacqui Mendez, M.S. October, 2012

Confidential and Proprietary. Not for Further Disclosure.

Presentation Overview

• Introduction to challenges of mammalian protein expression and analysis

• HaloTag Fusion Protein Technology

• How to get started and collection of pre-made clones

• Using the technology for:

• Cellular localization studies

• Protein purification

• Protein: nucleic acid analysis

• Protein: protein interaction studies

• Coupling with NanoLuc technology for BRET assays

• Summary

2


Current Technologies for Protein Analysis

3

Cell Based Analysis Localization, Trafficking

Biochemical and Proteomic Analysis Detection, Purification, Interactions

Antibodies

Affinity Tags

Fluorescent Proteins

A system applicable to the all approaches that also addresses limitations of current methods

Minimal interference with protein of interest Efficient capture/isolation High Signal/background

Detection Real-time imaging Differential labeling


What is HaloTag Technology? A Unique, Multifunctional Protein Fusion Tag

O Cl O Functional

Group

HaloTag

+

N- or C- terminal fusions

Chloroalkane Linker Protein of

Interest

Functional Group

Protein of Interest

O O

Covalent bond between HaloTag and chloroalkane (+ functional group)

4

HaloTag:

• Based on a 34.1 kDa monomeric halophilic bacterial hydrolase

• Engineered to covalently bind chloralkane substrates resulting in irreversible attachment

• Optimized for fast kinetics and stability.

• No homolog in mammalian cells = no background

Read more about the development of this

powerful fusion tag: Ohana, R.F., et al.

(2009) Prot. Exp. Purif. 68, 110-120.


Many Functional Groups are Available Same genetic construct can be used for multiple applications

Functional Group

Protein of Interest

O O

O Cl O

O Cl O

O Cl O

O Cl O

HaloTag Surfaces/Resins Capture and Display

Protein arrays Purification Interactions

HaloTag Fluorescent Ligands Labeling and Detection

Cellular imaging Gel analysis Quantitation

HaloTag Reactive Ligands Custom Modifications

Attach to particles, surfaces

Attach special ligands

Magnetic, non-magnetic resins, glass slides

Many colors of cell permeable &

impermeable ligands

e.g. Quantum Dots, PET ligands

5


Experimental Workflow to Understanding Protein Function Complementary studies to characterize global proteomics

Transfection/ Expression

HaloTag Technology enables & improves proteomics research

Purification

Cellular Localization

Cloning

Protein Interactions


Transfection/ Expression Purification


Cloning


Starting to Use HaloTag Technology Cloning Vectors and Premade Clones


Cloning your own ORF into HaloTag Vectors N- and C- terminus, variable expression levels

HaloTag Flexi Vectors for mammalian cell expression

Expression Level Promoter N-terminal HaloTag C-terminal HaloTag

Maximal expression CMV pFN21 pFC14

Promoter deletion series to optimize mammalian expression level

CMVd1 pFN22 pFC15

CMVd2 pFN23 pFC16

CMVd3 pFN24 pFC17

Flexi Vector Cloning System

• Flexible system for directional cloning utilizing rare cutter REs

• Efficient, simplified transfer to other Flexi vectors

• Sequence once & transfer


Premade HaloTag-ORF Clones Extensive collection validated clones

Kazusa DNA Research Institute & Promega

•Collaborating to create HaloTag-ORF expression clones

•8,882 clones available and increasing

• Unique catalog number for each clone

•N-terminal fusions in pFN21A Flexi vector

•Validated by: Sequence

Insert size

Expression validated in transfected HEK 293 cells by SDS-gel

In vivo expression verified by fluorescence microscopy

•Custom clones available

Validation Criteria Percent Validated Sequenced 100%

Insert size 99.98%

Expression - HEK 293/SDS-PAGE 99.4%

Expression - Fluorescence microscopy 77.91%


Premade HaloTag-ORF Collection Excellent Representation Across All Protein Classes

Kinases Tyrosine kinases MAP kinases G-Protein coupled receptors (G-PCRs) Phosphatases Transcription factor-related genes Membrane proteins Protein complexes

Cytokines & chemokines Ubiquitin hydrolases (DUBS) Ubiquitin ligases (E1, E2, E3) Tumor suppressors Oncogenes Transporters and pumps Proteases Epigenetics

Clones in many pathways focused on

transcription and important disease targets


Identify Your HaloTag-ORF Clone and More with “Find My Gene” & Click to Order

www.promega.com/FMG

•Detailed gene/protein information • Interacting protein partners

1

http://www.promega.com/FMG




Cloning


Cellular Localization Real time imaging, co-localization, trafficking


Intracellular Protein Labeling and Imaging Simple Labeling Protocols

Transfect HT-ORF expression vector

Add fluorescent ligand

GST

O O

Wash out unbound ligand

Image live or fixed cells

Alternate labeling protocol with no-washing

No cytotoxicity Minimal background fluorescence Multiple colors available

Cell permeable (complete cell staining) Cell impermeable (cell surface staining) Rapid and easy protocols


P

P

• HeLa cells expressing p65-HaloTag® labeled with TMR Ligand

• Treated with TNF

• Imaged (5min/frame; 120min)

NFkB Signaling Pathway

p50

p65

p50

p65

Signal (TNF, LPS, UV…)

Real-time Imaging

IkB

p50

p65

P

IkB p65

P

Nucleus Cytoplasm

p50

P

P

IkB p65

P p50

Cell membrane


Antibody POLR2A(CTD)

TMR HT-POLR2H

HaloTag-POLR2H

Overlay

Antibody

Y

Simultaneously confirm proper complex formation and localization

Add permeable HaloTag-TMR ligand

Image

• HaloTag-POLR2H is properly localized to the nucleus • Co-localization with antibody specific to the POLR2A CTD

Multiplexing HaloTag Imaging with Other Labeling Technologies


12hrs

Analysis of Protein Trafficking Spatial and Temporal Analysis of Localization

Svendsen, S., et al. BMC Cell Biol, 9: 17, 2008.

12 hours

1. Label with cell impermeable ligand

2. Label with cell permeable ligand

Label w/ impermeable GREEN

Label w/ permeable RED Wash Image Incubate

12 hrs Image

Detect proteins at

any given time

Differentiate

populations with

different color

ligands




Cloning


Purification and Detection Optimize expression and purify tag free protein

• Advantages of purification from mammalian cells

Native environment Proper folding, processing, and post-translational

modifications • challenges

Poor expression could cause low recovery and yield

Impurities


Purification

A better way of purifying mammalian proteins

Advantages of purification from mammalian cells

Native environment Proper folding, processing, and post-translational modifications

Challenges

Poor expression could cause low recovery and yield Impurities

HaloTag Selective & Covalent capture

Efficient protein capture even at low expression levels

No loss of bound protein during washes


Protein Purification from Mammalian cells Simple Protein Purification

Recovery Pure protein free of HT and

HaloTEV protease

Covalent Binding Proteolytic Release POI released by HaloTEV protease

HaloTEV & HT remain permanently attached to HaloLink

HaloLink

Resin

O

O

Protein of

Interest

HaloTag

O

O

POI TEV

O

O

POI


Purification of Human Kinases expressed transiently in HEK293T Cells

Ohana, R.F., et al. Prot. Exp. and Purif. (2011) 76, 154-64

Standard GST:HaloTag (pmols)

0.4 0.8 1.6 3.2 PKCg PKAc Src DEGFR PI3Kg

Protein standard and fusions were labeled with HaloTag TMRDirect ligand

Quantitative

detection

Purification


Purification of Human Kinases Highly Active Purified Proteins

*Kinase activity was assayed using ADP-Glo assay

Measured specific activities are in agreement with reported values

0

1,000,000

2,000,000

3,000,000

0 5 10 15

RFU

PRKCγ (ng)

Activity of purified PRKCγPKCtide PKCtide+ inhibitor (staurosporine)


Kinase Measured Specific Activity

(nmol/min/mg) Reported Specific Activity

(nmol/min/mg)

PKCg 16,551 2,260

PKAc 9,670 8,580

Src 1,624 1,032

DEGFR 196 101

PI3Kg 233 39


Comparison to Other Affinity Tags Greater Recovery, Yield, and Purity with HaloTag





Cloning


Protein Interactions Enabling the study of protein:DNA & protein:protein


HaloCHIP: Protein:DNA Chromatin Isolation without Antibodies

No Ab needed Consistent normalization between samples Analyze recovered DNA

End point PCR ChIP-chip arrays ChIP-seq


CREB HaloCHIP-chip Genome Wide Analysis

W.G.A./ Labeled DNA

10-50ng 1-10µg

HaloCHIP™ DNA

HT-CREB

Control

CREB HaloCHIP-chip Array

27,661 promoters - 385,000 probes TSS

~1.8kb

TSS

TSS

Uni-directional

Bi-directional

OR

Promoter Coverage

Hartzell, D.D., et al. BMC Genomics, 10: 497 (2009)


Gene Ontology (GO) and Promoter Analysis

Cellular Functions # of Promoters p-value

Histone Assembly 12/65 1.26E-06

Chromatin architecture 20/261 7.63E-07

Ribonucleic Complexes 26/392 7.06E-07

RNA processing 26/395 8.01E-07

DNA metabolism 38/638 2.93E-08

Nucleic acid binding 110/2764 2.19E-09

CREB HaloCHIP-chipTop 1% Promoters

• Identified CREB bound promoters all known to be regulated by CREB • Binding profile shows peaks of DNA binding above CRE consensus sites

Promoter Binding Profile

TSS

Half CRE

Full CRE



HaloCHIP and ChIP Binding Patterns Similar results with both approaches


Half CRE

Full CRE

TSS

Uni-directional Promoter

Half CRE

Full CRE

TSS TSS

Bi-directional Promoter

A direct comparison with the endogenous CREB protein show similar binding profiles for uni-directional and bi-directional promoters.


HaloTag Applications: Mammalian Pull-Downs Protein: Protein Interactions

28

TEV Cleavage

SDS/Urea Elution

POI

Halo Link

POI

HT

HaloTag fusion

construct

HT

POI

Analysis of protein complexes (new discovery or validation) • Mass spec • Western blotting

Halo Link HT


Expression Studies and Isolation of Human RNA Polymerases

Expression Levels of Halo-POLR2H

Fused HaloTag to H subunit

Pull-down Subunits of the RNA Polymerases

• A CMV deletion series yielded HT-POLR2H expression over a 50-fold range

• Pull-down performed for each in triplicate and analyzed by MudPIT mass spectrometry

Daniels et al J. Proteome Res. 2012, 11, 564-575.

CMV promoter deletion series

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Subunits shared among all RNAPs

RNAP II specific subunits RNAP III specific subunits

RNAP I specific subunits

Identification and quantitative proteomics analysis of isolated RNA Polymerase subunits

dNSAF values for subunits of isolated RNA Polymerase complexes High expression

Medium expression

Low expression

Ultralow expression

RNAP III specific subunits

• Consistent and reproducible capture of all core RNA Pol I, II and III subunits Daniels et al J. Proteome Res. 2012, 11, 564-575.


Known Interactors High Medium HT Ctrl

RPAP2 XXX XXX

RPAP3 XXX XXX

Elong F II X

PAF XX

CDC73 XXX XXX

XAB1 X X

RuvB-like 1 XXX XXX X

RuvB-like 2 XXX XXX XX

ELONGIN B XXX X

ELONGIN C X X

Novel Interactors MLLT11 XXX XXX

LOC85395 XXX XXX

LOC90488 XXX XXX

Identification of associated RNA Pol II factors and discovery of new interacting protein partners

• Several known associated factors were identified in the pull-down

• Three novel interactors were identified: MMLT11, LOC85395, LOC90488


Reciprocal complex isolation and mass spec analysis of identified interacting proteins

MLLT11

LOC85395

LOC90488

Reciprocal pull-down dNSAF values of RNA Pol II subunits dNSAF values of associated proteins

silver stain

• Most abundant proteins in all three pull-downs are known interacting partners of RNA Pol II

• Only a few core subunits of RNA Pol II were detected

• The novel proteins may be secondary or tertiary interactors of RNA Pol


HaloTag and NanoLuc BRET assay Live detection of Protein:Protein Interactions

1. Addition of luminescence

substrate

2. Addition of fluorescent ligand

NanoLuc Luciferase Donor

• 19kD, Monomeric • 100-150X brighter • N- or C-term fusion

HaloTag Fluorescent Acceptor

34kD, Monomeric Covalent labeling N- or C-term fusion

Energy transfer from bioluminescent donor to fluorescent acceptor resulting in emission of light

Enables real time monitoring of protein-protein interaction in vivo Using HaloTag and NanoLuc technologies, BRET can be achieved with increased

signal and decreased spectral overlap

Focus on epigenetics and interactions with chromatin using HaloTag Histones as the acceptor proteins

NanoLuc Donor

HaloTag® Acceptor

NanoLuc® Fusion Protein

HaloTag® Fusion Protein


Live Cell Imaging of HaloTag Histones Testing proper incorporation into chromatin

H2A-HaloTag

H2B-HaloTag

H3.3-HaloTag

H4-HaloTag

Scale bars = 10µm

HaloTag fusion

HT

Histone

TMR

Confocal Imaging

Observed chromatin incorporation of HaloTag fusions: H2A, H2B, H3.3, and H4

34


BRET Assay Detects BRD4 Interactions with Histones

35

Detected NLuc-BRD4 interaction Halo-Histones

HaloTag fusion

HT

H3.3, H4 NanoLuc fusion

NanoLuc Luciferase

BRD4

BRET (600/450nm) Measurement

TMR EmissionMAX=585

Furimazine EmissionMAX = 460

0.0060 0.0054

0.0000

0.0010

0.0020

0.0030

0.0040

0.0050

0.0060

0.0070

0.0080

Histone H3.3 Histone H4A

vera

ge 6

00

/45

0 B

RET

Rat

io

BRET measurement in HCT 116 cells


Measuring Effect of BRD4 Inhibitor on Histone H3 or H4-Bromodomain Interactions

IC50 = 65.4 nM

IC50 = 222.8 nM

Histone H4-HaloTag

Measured potency

differences amongst

bromodomain proteins and

histones

Histone H3.3-HaloTag

NLuc-BRD4

NLuc-BRD4 NLuc-CBP

NLuc-CBP

36


Summary HaloTag technology

37

HaloTag provides a multifunctional handle on your protein of interest and its unique capabilities enables improved proteomics research.

An extensive collection of pre-made clones is available.

A variety of applications for functional proteomics studies:

Monitor cellular localization both spatially and temporally

Simple and efficient mammalian protein purification

Analyze protein: DNA interactions in vivo (HaloCHIP)

Highly specific capture of protein complexes allow discovery and validation of protein: protein interaction studies

Monitor in vivo interactions and the effect of inhibitors with BRET assays


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By phone: 800-356-9526

•Available 7am-6pm MST M-F

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•Available 7am-6pm CST M-F

•Global Chat with Branch office tech serv scientists, too, after hours (language dependent)

By email: [email protected]

•Guaranteed answers within 24hr

•Most responses within 2hrs

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accelerating proteomics research with a unique, multi

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