Download What is a yeast two hybrid assay? How has this approach been

What is a yeast two hybrid assay?
How has this approach been used
to look globally or genome-wide at
protein-protein interactions? What
is a one hybrid assay?
Jamie Feldmann
Shweta Lahudkar
John Martin
Yeast two hybrid assay
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Performed in S. cerevisiae most commonly
Binding Domain
Activating Domain
Interact without direct binding
Yeast two hybrid assay
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Reporter transcript
Known protein as bait
cDNA library
If interact, reporter transcript activated
Positive vs Negative Selection
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Summary of
process
How has this approach been used to
look globally or genome-wide at
protein-protein interactions
Yeast two hybrid assay
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Functions of new found proteins inferred
through interactions with known proteins
Expected interactions Æ what reacts with
known proteins
By mutating amino acids, can determine
importance of those remaining
zFig.
2: General flowchart
of a hypothetic two-hybrid
screen. Pointed lines should
be followed in case of negative
results
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Yeast Two-Hybrid: State of the Art Wim Van
Criekinge1* and Rudi Beyaert2
1 DEVGEN NV. Technologiepark 9, 9052
Zwijnaarde-Gent. Belgium. Phone: 32-9-264-5403 Fax: 32-9-264-54-18
2 Department of Molecular Biology. Flanders
Interuniversity Institute for Biotechnology and
University of Ghent, B-9000, Ghent. Belgium.
What is a one hybrid assay?
One hybrid assay
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Used for protein-DNA interactions
AD is directly bound to BD
BD not of fixed sequence, may be a library
Disadvantages
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Known sequence for AD or BD
Need a protein of known function
Have to assume each cell only takes up one
plasmid
Fusion might change shape of bait protein
Interpreting significance
Advantages
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Can be done in vivo
One plasmid is sufficient
Only need indirect interaction
Yeast is host
Thank You!!