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The Yeast Two-Hybrid System

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Title: The Yeast Two-Hybrid System


1
The Yeast Two-Hybrid System
  • Anne C. Luebke

2
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)

3
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

4
The Model
Transcription Activating Region
Bait Protein
Prey Protein
DNA-Binding Domain
Reporter Gene
DNA-Binding Site
5
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

6
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

7
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)

8
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

9
Sample Plasmid
From Golemis Lab Homepage
10
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)

11
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

12
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

13
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

14
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.

15
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
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