The Yeast Two-Hybrid System

Presentation Transcript

Anne C. Luebke

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)

Transcription

Activating

Region

Bait Protein

Prey Protein

DNA-Binding

Domain

Reporter Gene

DNA-Binding Site

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

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

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)

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

From Golemis Lab Homepage

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)

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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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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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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