Yeast two hybrid system / protein-protein interaction
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Transcript of Yeast two hybrid system / protein-protein interaction
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Presented To : Dr. AmberPresented By: Maryam Shakeel
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It is a large scale study of proteins, particularly their structures and functions.
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Y2H is a molecular biology technique
In vivo technique Protein-protein interaction
Premise
Transcriptional activation
Reporter gene
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1. Two Domains of Transcriptional activators
Transcriptional Activator
DNA Binding Domain
Activating Domain
Recognize specific sequence
Enable RNA Polymerase to
transcribe reporter gene
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Gal4
X
Y z
X = Bait ProteinY= Pray Protein
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Two hybrid proteins are generated with transcription factor domains
Interaction between Bait and Prey is necessary for transcription.
Reporter Gene
X
Binding
Domain
Y
Activation
Domain
UAS
X transcription
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The reporter gene use in Gal4 system is LacZgene.
Reporter Gene
X
Binding
Domain
Y
Activation
Domain
UAS
Protein-protein interaction can be measure by measuring transcription of reporter gene.
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Bait Plasmid
Prey Plasmid
TRP use as selectable marker
Leu used as selectable marker
Prey Plasmid
Bait Plasmid
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Strain carry mutations in different genes for A.A biosynthesis such as TRP1, LEU2, HIS3 and URA3.
Grow in growth medium lack these A.A Only hybrid plasmid carrying transformant
yeast grow.
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1. Highly sensitive 2. Elegant mean to study protein-protein
interaction.3. Characterize these interaction
4. A fairly new addition to the family of microbiological studies
Which protein domain is
responsible
Conditions for interaction
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Limited to those proteins
Transcription occurs only when DBD bound to AD
Localize in nucleus
Stable in yeast cell
Continue its activity in yeast cell
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Protein-Protein Interactions: Methods and Applicationsedited by Haian Fu
The Yeast Two-hybrid Systemedited by Paul L. Bartel, Stanley Fields
http://labrat.fieldofscience.com/2010/01/exploring-protein-interactions-yeast.html
http://www.hybrigenics-services.com/contents/resources/yeast-two-hybrid-principle
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