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Efficient isolation and identification of intracellular protein complexes from mammalian cells using HaloTag® technology Poster # 331Jacqui Méndez, Nancy Murphy, Danette D. Hartzell, Natasha Karassina, Georgyi Los, Marjeta Urh and Keith WoodPromega Corporation, 2800 Woods Hollow Road, Madison, WI 53711 [email protected]
1. Introduction
2. HaloTag® Technology
6.
3. In vivo HaloTag Pull-Down Protocol
7. Summary
http://www.promega.com/applications/prtn_exp/interactions.htm
http://www.promega.com/halotag
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.
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.
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.
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.
Beyond complex isolation: Studying different aspects of the NFκB pathway with one construct
Verma and Stevenson, 1997
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.
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).
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.
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 multi-protein 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).
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).
OO Functional group
HaloTag is a protein fusion tag engineered to covalently bind a synthetic chloroalkane ligand.
Irreversible attachment to a series of functional groups impart variable chemical functionalities:
Attachment to surfaces:Interaction analysisProtein purificationImmobilization
Attachment to fluorophores:Cellular imagingQuantificationGel analysis
OO
OO
1. Expression of HaloTag fusion and formation of complexes .
2. Cell lysis , complex capture, and washing.
3. SDS elution OR TEV cleavage to release capture proteins.
4. Downstream analysis by mass spectroscopy or Western blot.
Transfection/Stable ExpressionHaloTag®-
Vector
HaloTag
POI
HaloTag alone (no POI) OR un-transfected cells
in parallel reaction as negative control
POI
HT
SDS
4. Isolation of the multi-protein Nup107-160 complex
5. Capture of the members of the NFκB pathway Protein of
Interest
HaloTag
POI
Resin
POI
HT
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.
p105 p105
p50
IκB-HT HT (-) Control
p65
p50
p65-HT HT (-) Control
IκB
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).
Bapteste et al., 2005
Nup37-HT Nup43-HT HT (-) Control
NUP 160NUP 133NUP 107
NUP 98/96NUP 85/Seh 1
NUP 43
NUP 160NUP 133NUP 107
NUP 98/96NUP 85/Seh 1
NUP 37
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.
Protein ofInterest
HaloTag
Protein ofInterest
HaloTag
CaptureHaloLink™
Resin
OO OO
LabelingFluorescent
ligands
OO
(A)Protein:protein
0 30 60 90 min. TNF
Western – Anti-I B
0 15 30 45 60
PCR – I B promoter
min. TNF(B)
Protein:DNA(HaloCHIP™)
15 minNucleus
0 15 30 110
Fluorescent TMR ligand
min. TNF
(C)Localization
HTp65
HTp65
120100
80
60
50
40
p105p100
p65
IkBα, IkBβ, IkBε
p65-HT HT (-) Control
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.
