Yeast 2-Hybrid2.26.18

Kayla Nygaard

Y2H -What is it?

A method to screen

for protein-protein interactions

in yeast

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

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

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

Y2H: Possible Outcomes

Stop & Jot

▪List 5 essential components in the

Yeast 2-Hybrid assay.

▪30 seconds – go!

Y2HVisual

Y2H: Reporters

HIS3 – Allows growth in the presence of HIS3 inhibitor 3AT

LacZ – turns colonies blue in the presences of X-gal

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

Y2H: Outcomes

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

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

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)

How to Y2H:Step 4

▪ Grow bacteria

▪ Harvest plasmid(s)

▪ Transform into yeast

How to Y2H:Step 5

▪ Perform Screen

Replica Plating

Think About It

▪ Is there any way you could use a

Y2H assay in your proposal?

▪Why/why not?

Y2H: OptionsTraditional Assay

http://mmg-233-2013-genetics-genomics.wikia.com/wiki/Yeast_Two-hybrid

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

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

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

Met

hods

http://mmbr.asm.org/content/76/2/331/F5.large.jpg

Other versions of Y2H

▪ Split-ubiquitin system to conquer membrane-bound proteins

▪ CUB = c-terminal; NUB = n-terminal

Other versions of Y2H

▪ Incorporating co-factors:

Applications

▪ Determining sequences crucial for interaction

▪ Drug and Poison Discovery

▪ Determination of protein function

▪ Zinc finger protein selection

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

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