Download Efficient Isolation and Identification of Intracellular Protein

Efficient isolation and identification of intracellular protein complexes from mammalian cells using
HaloTag® technology
Poster # 331
Jacqui Méndez, Nancy Murphy, Danette D. Hartzell, Natasha Karassina, Georgyi Los, Marjeta Urh and Keith Wood
Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711 U.S.A.
[email protected]
1. Introduction
4. Isolation of the multi-protein Nup107-160 complex
 HaloTag (HT) fusion proteins form a highly specific and covalent bond with the
HaloLink resin allowing rapid capture of dilute protein complexes from cellular lysates
in a single step purification method.
Nup37-HT
Nup43-HT HT (-) Control
HaloLink™
Resin
p65
 The HaloTag technology is also applicable for the study of in vivo Protein:DNA
interactions and cellular localization of the protein of interest using fluorescent HaloTag
ligands.
 Irreversible attachment to a series of functional
Halo
Tag
Halo
Tag
Protein of
Interest
O
Using either NUP 37-HT or NUP 43 -HT six
components of the Nup107-160 NPC are
specifically isolated as determined by
mass spectroscopy analysis of the
recovered complex mixture.
5. Capture of the members of the NFκB pathway
O
NFκB are a family of structurally related nuclear transcription
factors involved in a number of cellular processes including
immune and inflammatory responses, cellular growth, and
apoptosis.
 Attachment to surfaces:
Interaction analysis
Protein purification
Immobilization
O
In the absence of stimuli p65 is primarily located in the
cytoplasm where it binds p50 (or its precursor p105 ); p52 (or its
precursor p100), and a number of IκB proteins (inhibitors of κB).
 Attachment to fluorophores:
Cellular imaging
Quantification
Gel analysis
O
In the presence of stimulation (such as with the tumor necrosis
factor TNFα) the IκBs are marked for degradation allowing the
p65/p50 dimer to move to the nucleus and activate transcription
of relevant genes such as IκB thus continuing the cycle.
Verma and Stevenson, 1997
3. In vivo HaloTag Pull-Down Protocol
HaloTag®Vector
POI
HaloTag
1. Expression of HaloTag
fusion and formation of
complexes .
HT
POI
HaloTag alone (no POI)
OR un-transfected cells
in parallel reaction
as negative control
2. Cell lysis , complex
capture, and washing.
Resin
HT
p105
p65-HT HT (-) Control
IκB-HT HT (-) Control
p65-HT HT (-) Control
Transfection/
Stable Expression
p105
120
100
80
p50
p50
p105
p100
p65
60
50
IκB
p65
40
IkBα, IkBβ, IkBε
POI
3. SDS elution OR TEV
cleavage to release
capture proteins.
SDS
POI
4. Downstream analysis by
mass spectroscopy or
Western blot.
O
O
60 90 min. TNF
Using either p65-HTor IκB-HT as
independent baits and HT alone as negative
(-) control the expected remaining protein
members of the NFκB cytoplasmic complex
are specifically isolated as confirmed by
Western blot analysis.
In this example using p65-HT as bait
the isolated protein partners can be
identified using mass spectroscopy
analysis from either individual gel
bands OR the entire complex mixture
in solution.
http://www.promega.com/applications/prtn_exp/interactions.htm
http://www.promega.com/halotag
p65
HT
O
(A)
Protein:protein
O
Labeling
groups impart variable chemical functionalities:
Protein of
Interest
O
Fluorescent
ligands
Bidirectional transport of macromolecules
between the cytoplasm and the nucleus
occurs through supramolecular structures
embedded in the nuclear envelope known
as nuclear pore complexes (NPCs).
 HaloTag is a protein fusion tag engineered to
covalently bind a synthetic chloroalkane ligand.
Functional
group
O
30
Western – Anti-I B
0
Bapteste et al., 2005
2. HaloTag® Technology
O
HT
NUP 160
NUP 160
NUP 133
NUP 133
NUP 107
NUP 107
NUP 98/96
NUP 98/96
NUP 85/Seh 1 NUP 85/Seh 1
NUP 37
NUP 43
 The ability to perform these different experiments with the same fusion protein
eliminates the need to make multiple constructs for each desired study allowing
flexibility to easily expand to other areas of research.
Halo
Tag
0
Capture
 Captured protein partners can be eluted using SDS or cleaved from the resin using
TEV protease and analyzed by mass spectrometry for the identification of unknown
binding partners or by Western blotting for the confirmation of known or suspected
protein partners.
Protein of
Interest
6. Beyond complex isolation: Studying different
aspects of the NFκB pathway with one construct
15
30 45 60 min. TNF
PCR – I B promoter
0
15
30
(B)
Protein:DNA
(HaloCHIP™)
15 min
Nucleus
110 min. TNF
(C)
Localization
O
Fluorescent TMR ligand
 Cells expressing p65-HT were stimulated with TNFα and samples at various
time points were either immobilized onto HaloLink resin to evaluate
Protein:Protein (A) and Protein:DNA interactions (B) or alternatively
fluorescently labeled to look at cellular localization (C).
 In the presence of TNFα there is a temporal correlation between the
amount of IκB protein bound to p65 recovered in the Protein:Protein study
(A); the amount of IκB promoter DNA recovered in the Protein:DNA
interaction analysis (B); and the cellular localization of the p65 protein (C).
7. Summary
 The HaloTag technology provides a convenient one step purification
method for the isolation of in vivo multi-protein complexes from
mammalian cells (3).
 The HaloTag Pull-Down method is capable of isolating large multiprotein structural complexes such as the NPC 107-160 (4) as well as
smaller regulatory protein complexes such as the NFκB complex (5).
 Recovered protein partners can either be analyzed by Western blotting
if binding partners are known or by mass spectroscopy when
discovering novel interactions. Furthermore, mass spectroscopy
analysis can be done from individual protein bands or from complex
in-solution mixtures (5).
 The versatility of the HaloTag technology allows a multitude of assays
to be performed from the same construct including detection of
Protein:Protein interactions; Protein:DNA interaction studies; and real
time intracellular protein imaging (6).