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1. 1
Identification of Protein-Protein Interactions by
Yeast 2-Hybrid System & Immunoprecipitation

2. Protein-Protein interactionsProtein-Protein interactions
 Important field in cell biologyImportant field in cell biology && biochemistrybiochemistry
 Localization and traffickingLocalization and trafficking
 Posttranslational modificationsPosttranslational modifications
 Signaling networksSignaling networks
 Important field in viral replication very difficult to predictImportant field in viral replication very difficult to predict
 two main patterns:two main patterns:
■■ domain-domain interactionsdomain-domain interactions
■■ domain-peptide interactionsdomain-peptide interactions
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4. Co-immunoprecipitation protocolCo-immunoprecipitation protocol
 Protein A binds to antibodiesProtein A binds to antibodies
 Sepharose beads coated with protein ASepharose beads coated with protein A
 Specific antibody binds to the protein of interestSpecific antibody binds to the protein of interest
 The complex is precipitated by binding to the beads viaThe complex is precipitated by binding to the beads via
protein Aprotein A
 Proteins are released from beads by boilingProteins are released from beads by boiling
 Analysis by SDS-PAGE and/or other methodsAnalysis by SDS-PAGE and/or other methods
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6. Detection of protein interactions by microarraysDetection of protein interactions by microarrays
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7. Principle of protein microarray technologyPrinciple of protein microarray technology
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8. Reporter Gene
Bait
Protein
Binding
Domain
Prey
Protein
Activation
Domain
The yeast two-hybrid systemThe yeast two-hybrid system……
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 Two hybrid proteins are generated with transcriptionTwo hybrid proteins are generated with transcription
factor domainsfactor domains
 Both fusions are expressed in a yeast cell that carries aBoth fusions are expressed in a yeast cell that carries a
reporter gene whose expression is under the control ofreporter gene whose expression is under the control of
binding sites for the DNA-binding domainbinding sites for the DNA-binding domain

9. Reporter Gene
Bait
Protein
Binding
Domain
Prey
Protein
Activation
Domain
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 Interaction of bait and prey proteins localizes the activationInteraction of bait and prey proteins localizes the activation
domain to the reporter gene, thus activating transcription.domain to the reporter gene, thus activating transcription.
 Since the reporter gene typically codes for a survivalSince the reporter gene typically codes for a survival
factor, yeast colonies will grow only when an interactionfactor, yeast colonies will grow only when an interaction
occurs.occurs.

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21. Bait SelectionBait Selection
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 Proteins known to be expressed in B cells and/or cardiacProteins known to be expressed in B cells and/or cardiac
myocytes.myocytes.
 Proteins known or suspected to be involved in signalingProteins known or suspected to be involved in signaling
pathways in B cells and/or cardiac myocytes.pathways in B cells and/or cardiac myocytes.
 Proteins believed important in GPCR or BCR signalingProteins believed important in GPCR or BCR signaling
leading to PIP3 generation.leading to PIP3 generation.

22. Bait DesignBait Design
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1-100 (P sites)
100-200 (Pro regions)
1-240 (N-term)
150-350 (linker)
348-457 (SH2)
210-457 (C-term)
110 116 134 146 350 436
P P P SH2Pro Pro
Phosphorylation
sites
Proline-rich
SH3 binding Src homology 2 domain
BLNK

23. B Cell Receptor PathwayB Cell Receptor Pathway
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24. B Cell Receptor PathwayB Cell Receptor Pathway
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25. BB Cell Receptor PathwayCell Receptor Pathway
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Pak
Nck
Pix/Cool
Git2
14-3-3z
Endophilin2
BAN-P
PDE4B3
Ruk1
-
PP2C
gamma

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30. CD19CD19
Actin
Cytoskeleton
PDK1PDK1
PI3Kγ (p110)PI3Kγ (p110)
BtkBtk
WISHWISH
CAPCAP
DblDbl
CamK IICamK II
NdkBNdkB
cdc42cdc42
CD22CD22
FynFyn
SOS2SOS2
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31. CD19CD19
BtkBtk
Actin
Cytoskeleton
SOS2SOS2
WISHWISH
CAPCAP
DblDbl
CamK IICamK II
NdkBNdkB
cdc42cdc42
PDK1PDK1
CD22CD22
FynFyn
PI3Kγ (p110)PI3Kγ (p110)
1907019719070197
67553996755399
66715386671538
46335144633514
85673258567325
1354267713542677
2089443020894430
2632696826326968
30642623064262
Sam68
Protein 4.1G
AIP
Cbl-b
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33. ReferencesReferences
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 Bonifacino JS, Dell’Angelica EC, Springer TA. 2001. Immunoprecipitation.Bonifacino JS, Dell’Angelica EC, Springer TA. 2001. Immunoprecipitation.
Curr. Protoc. Mol. Biol. Chapter 10:Unit 10.16Curr. Protoc. Mol. Biol. Chapter 10:Unit 10.16..
 Protein Microarray techniques. S. Funke Dipl.Biol. Universitäts-Protein Microarray techniques. S. Funke Dipl.Biol. Universitäts-
Augenklinik Mainz. Leitung: PD Dr. Dr. F. Grus. Langenbeckstr. 1, 55131Augenklinik Mainz. Leitung: PD Dr. Dr. F. Grus. Langenbeckstr. 1, 55131
MainzMainz
 Yeast Two-Hybrid, a Powerful Tool for Systems Biology. Anna Brückner,Yeast Two-Hybrid, a Powerful Tool for Systems Biology. Anna Brückner,
Cécile Polge, Nicolas Lentze, et al. Int. J. Mol. Sci. 2009, 10, 2763-2788;Cécile Polge, Nicolas Lentze, et al. Int. J. Mol. Sci. 2009, 10, 2763-2788;
doi:10.3390/ijms10062763doi:10.3390/ijms10062763