Yeast 2-Hybridgenetics.wustl.edu/bio5491/files/2018/02/KRN-Yeast-2-Hybrid.pdf · Reverse Yeast...
Transcript of Yeast 2-Hybridgenetics.wustl.edu/bio5491/files/2018/02/KRN-Yeast-2-Hybrid.pdf · Reverse Yeast...
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Yeast 2-Hybrid2.26.18
Kayla Nygaard
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Y2H -What is it?
A method to screen
for protein-protein interactions
in yeast
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How does it work?
▪ Capitalizes on GAL4 system in yeast.
▪ GAL4 has 2 domains
▪ DNA-Binding Domain (DB)
▪ Transcriptional Activation Domain (AD)
https://www.sciencedirect.com/science/article/pii/B9780124201200000037
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How does it work?
▪ These domains can be separated & fused to
proteins of interest (X, Y)
▪ DB-X = bait
▪ AD-Y = prey
https://www.sciencedirect.com/science/article/pii/B9780124201200000037
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How does it work?
▪ If X & Y interact, GAL4 domains will reunite
and activate a reporter gene.
https://www.sciencedirect.com/science/article/pii/B9780124201200000037
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Y2H: Possible Outcomes
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Stop & Jot
▪List 5 essential components in the
Yeast 2-Hybrid assay.
▪30 seconds – go!
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Y2HVisual
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Y2H: Reporters
HIS3 – Allows growth in the presence of HIS3 inhibitor 3AT
LacZ – turns colonies blue in the presences of X-gal
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Caveats
▪ Learn as much as possible about X and Y
▪ Is the bait protein (X) known to activate transcription
(or repress it?)
▪ Does bait or prey belong to a protein family?
▪ When & where are they expressed
▪ Will be necessary in construct cDNA library
▪ How will you confirm interactions from screen?
▪ Co-IP of endogenous proteins
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Y2H: Outcomes
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How to Y2H: Step 1
CONSTRUCT DB FUSION PROTEIN
▪ Clone bait DNA fragment into plasmid
▪ pDBLeu = GAL4 DNA-binding domain vector
▪ Via restriction enzyme digest + ligation
▪ May need to amplify bait DNA fragment with primers
containing correct restriction sites
▪ Transform into competent E. coli
▪ Plate on kanamycin plates for selection
▪ Sequence at junction to confirm correct reading frame
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How to Y2H: Step 2
CONSTRUCT AD FUSION
PROTEIN
▪ Clone prey DNA fragments into plasmid
▪ pPC86 = GAL4 activation domain vector
▪ Same process as DB fusion
▪ Transform
▪ Plate on ampicillin plates
▪ Sequence
▪ **usually a library of cDNA products
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How to Y2H: Step 3
*TEST FOR SELF-ACTIVATION
▪ Measure HIS3 basal expression due to self-activation
▪ In both strains (pDBLeu-X and pPC86-Y)
▪ Increase 3AT in yeast media plates w/ histidine
▪ 3AT inhibits HIS3 in dose-dependent manner
▪ Titrate HIS3 activity until growth is inhibited
▪ Determine minimum effective dose
▪ Allows for weak protein-protein interactions to be detected
▪ For best results test with AD vector (minus prey)
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How to Y2H:Step 4
▪ Grow bacteria
▪ Harvest plasmid(s)
▪ Transform into yeast
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How to Y2H:Step 5
▪ Perform Screen
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Replica Plating
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Think About It
▪ Is there any way you could use a
Y2H assay in your proposal?
▪Why/why not?
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Y2H: OptionsTraditional Assay
http://mmg-233-2013-genetics-genomics.wikia.com/wiki/Yeast_Two-hybrid
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Advantages
▪ Can detect transient interactions that might be missed
in co-IP
▪ Semi-quantitative
▪ Can be used to study known interactions
▪ Modify residues, check if interaction is maintained
▪ Immediate identification of the gene that encodes the
product
▪ By sequencing the plasmid from the colony of interest
▪ Can be scaled up easily or automated
▪ Low-tech – no sophisticated equip. necessary
▪ Important first hint at interactions
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Potential Weaknesses
▪ False positives
▪ Proteins may not be expressed together normally
▪ Unnatural concentrations
▪ Non-specific interactions
▪ False negatives
▪ Proteins may not localize to the nucleus
▪ Fusion to AD or BD may block
▪ Interaction may not occur in yeast
▪ May lack necessary chaperones so proteins unfolded
▪ Protein may miss necessary phosphorylation
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Y2H Adaptations
▪ Reverse Yeast 2-Hybrid
▪ Detect when an interaction is disrupted
▪ Yeast 3-hybrid
▪ Detect protein-RNA interactions
▪ Yeast 1-hybrid
▪ Detect protein-DNA interactions
▪ Split-ubiquitin assay
▪ To detect membrane-bound protein interactions
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Met
hods
http://mmbr.asm.org/content/76/2/331/F5.large.jpg
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Other versions of Y2H
▪ Split-ubiquitin system to conquer membrane-bound proteins
▪ CUB = c-terminal; NUB = n-terminal
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Other versions of Y2H
▪ Incorporating co-factors:
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Applications
▪ Determining sequences crucial for interaction
▪ Drug and Poison Discovery
▪ Determination of protein function
▪ Zinc finger protein selection
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Other model systems
▪ S. cerevisiae = original yeast used
▪ Candida albicans
▪ For C. albicans genes
▪ E. coli = B2H
▪ Use larger libraries, lower false positives
▪ Mammalian two-hybrid = M2H
▪ Uses transfected cell lines
▪ Arabidopsis thaliana = P2H
▪ For plants! (protoplast two hybrid = P2H)
▪ Bombyx mori = I2H
▪ Insect two-hybrid
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HW
▪Will be posted later today.
▪Read related papers if you haven’t yet.
▪Help with Dutcher HW today at 5pm in
6th McKinley/Couch