The Yeast Two-Hybrid System

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The Yeast Two-Hybrid System

• Anne C. Luebke

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What is the yeast two-hybrid system used for?

• Identifies novel protein-protein interactions
• Can identify protein cascades
• Identifies mutations that affect protein-protein
  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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The Model
Transcription Activating Region
Bait Protein
Prey Protein
DNA-Binding Domain
Reporter Gene
DNA-Binding Site
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Steps to Screen a Library

• 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

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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
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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

• 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.
• 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.
• Fields, Stanley, O. Song. "A novel genetic
  system to detect protein-protein interactions."
  Nature (1989) Vol. 340, p.245-246.
• 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.
• 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.
• 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.
• 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.

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References continued

• 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. 10321-10326.
• White, Michael. "The yeast two-hybrid system
  Forward and reverse." Proc. Natl. Acad. Sci. USA
  (1996) Vol 93, p. 10001-10003.
• 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.
• Lab of Erica Golemis http//www.fccc.edu/research/
  labs/golemis/EG_homepage.html
• Special thanks to Dr. Susan Mango and the
  University of Utah