Transcript General

The Yeast Two-Hybrid System
Anne C. Luebke
What is the yeast two-hybrid
system used for?
Identifies novel protein-protein interactions
 Can identify protein cascades
 Identifies mutations that affect proteinprotein binding
 Can identify interfering proteins in known
interactions (Reverse Two-Hybrid System)
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How does it work?
Uses yeast as a model for eukaryotic protein
interactions
 A library is screened or a protein is
characterized using a bait construct
 Interactions are identified by the
transcription of reporter genes
 Positives are selected using differential
media
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Transcription
Activating
Region
The Model
Bait Protein
DNA-Binding
Domain
DNA-Binding Site
Prey Protein
Reporter Gene
Steps to Screen a Library
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Create the Bait Plasmid Construct from the gene
of interest and the DNA binding domain of Gal4
or LexA or other suitable domain
Transform with the bait construct a yeast strain
lacking the promoter for the reporter genes and
select for transformed yeast
Transform the yeast again with the library
plasmids
Select for interaction
Sequence analysis
Isolate plasmid from yeast and transform E.
coli
 Purify plasmid from E. coli and sequence
 Blast sequence against database for known
proteins or construct a possible protein
sequence from the DNA sequence and
compare to other proteins
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Reporter Genes
LacZ reporter - Blue/White Screening
 HIS3 reporter - Screen on His+ media
(usually need to add 3AT to increase
selectivity)
 LEU2 reporter - Screen on Leu+ media
 ADE2 reporter - Screen on Ade+ media
 URA3 reporter - Screen on Ura+ media (can
do negative selection by adding FOA)
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Plasmid Constructs
Plasmids are constructed with the Gal4
DNA binding domain (or other suitable
domain) in front of a Multiple Cloning Site
(MCS)
 The plasmid contains genes that can be used
for selection such as Amp, Leu2, Ura3, or
Trp1
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Sample Plasmid
From Golemis Lab Homepage
False Positives
False positives are the largest problem with
the yeast two-hybrid system
 Can be caused by:
 Non-specific binding of the prey
 Ability to induce transcription without
interaction with the bait (Majority of false
positives)
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Elimination of False Positives
Sequence Analysis
 Plasmid Loss Assays
 Retransformation of both strain with bait
plasmid and strain without bait plasmid
 Test for interaction with an unrelated protein
as bait
 Two (or more) step selections
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Advantages
Immediate availability of the cloned gene of
the interacting protein
 Only a single plasmid construction is
required
 Interactions are detected in vivo
 Weak, transient interactions can be detected
 Can accumulate a weak signal over time
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Examples of Uses of the Yeast
Two-Hybrid System
Identification of caspase substrates
 Interaction of Calmodulin and L-Isoaspartyl
Methyltransferase
 Genetic characterization of mutations in
E2F1
 Peptide hormone-receptor interactions
 Pha-4 interactions in C. elegans
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References
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Bartel, Paul, C. Chien, R. Sternglanz, S. Fields. “Elimination of False Positives that Arise in Using the Two-Hybrid
System.” Biotechniques (1993) Vol. 14, no. 6, p. 920-924.
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Chien, Cheng-ting, P. Bartel, R. Sternglanz, S. Fields. “The two-hybrid system: A method to identify and clone genes
for proteins that interact with a protein of interest.” Proc. Natl. Acad. Sci. USA (1991) Vol. 88, p. 9578-9582.
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Fields, Stanley, O. Song. "A novel genetic system to detect protein-protein interactions." Nature (1989) Vol. 340,
p.245-246.
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James, Philip, J. Halladay, E. Craig. "Genomic Libraries and a Host Strain Designed for Highly Efficient Two-Hybrid
Selection in Yeast." Genetics (1996) Vol. 144, p. 1425-1436.
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Kamada, S, H. Kusano, H. Fujita, M. Ohtsu, R. Koya, N. Kuzumaki, Y. Tsujimoto. "A cloning method for caspase
substrates that uses the yeast two-hybrid system: Cloning of the antiapoptotic gene gelsolin." Proc. Natl. Acad. Sci.
USA (1998) Vol 95, p. 8532-8537.
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O'Connor, Mirriam, C. O'Connor. "Complex Interactions of the Protein L-Isoaspartyl Methyltransferase and
Calmodulin Revealed with the Yeast Two-hybrid System." The Journal of Biological Chemistry (1998) Vol. 273, p.
12909-12913.
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Staudinger, Jeff, J. Zhou, R. Burgess, S. Elledge, E. Olson. "PICK1: A Perinuclear Binding Protein and Substrate for
Protein Kinase C Isolated by the Yeast Two-Hybrid System." The Journal of Cell Biology (1995) Vol. 128, p. 263-271.
References continued
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Vidal, Marc, P. Braun, E. Chen, J. Boeke, E. Harlow. "Genetic Characterization of a mammalian protein-protein
interaction domain by using a yeast reverse two-hybrid system." Proc. Natl. Acad. Sci. USA (1996) Vol. 93, p. 1032110326.
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White, Michael. "The yeast two-hybrid system: Forward and reverse." Proc. Natl. Acad. Sci. USA (1996) Vol 93, p.
10001-10003.
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Zhu, Jianwei, C. Kahn. "Analysis of a peptide hormone-receptor interaction in the yeast two-hybrid system." Proc.
Natl. Acad. Sci. USA (1997) Vol. 94, p. 13063-13068.
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Lab of Erica Golemis http://www.fccc.edu/research/labs/golemis/EG_homepage.html
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Special thanks to Dr. Susan Mango and the University of Utah