Yeast Two-Hybrid Assay to Determine Protein Self-Association in Yeast Cells

Samenvatting

Source: Stanczyk, P. J., et al., Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study. J. Vis. Exp. (2016)

This video demonstrates the yeast two-hybrid assay to detect specific protein self-interactions. In this assay, the protein of interest is tagged to two domains of a transcription factor that encodes the β-galactosidase enzyme. When the two proteins interact, this causes transcription and translation to form the enzyme, which is then detected using a biochemical assay.

Protocol

1. Yeast Transformation

1. Harvest yeast by centrifugation (in 50 ml tubes) at 1,500 x g for 5 min at RT. Discard the supernatant, resuspend each pellet in 5 ml of sterile H₂O, and pool together.
2. Re-centrifuge at 1,500 x g for 5 min at RT and discard the supernatant. Resuspend yeast pellet in 1 ml of freshly prepared, sterile 1x LiAc/TE.
   NOTE: Use competent yeast cells within 1 hr of preparation.
3. Prepare plasmid samples (in 1.5 ml tubes) by adding 200 ng of plasmid DNA for single transformations, or 0.5 - 1 µg of each plasmid DNA for co-transformations, and 100 µg of herring testes carrier DNA (boiled for 20 min and cooled on ice just prior to use).
    NOTE: Include a positive control, e.g., yeast co-transformed with pVA3 (encoding for GAL4 DNA-BD fusion with p53 protein) and pTD1 (encoding for GAL4 AD fusion with SV40 large T antigen).
4. Add 100 µl of the freshly prepared, competent yeast suspension and 600 µl of 1x LiAc/PEG solution (8 ml of stock PEG 3350, 1 ml of stock TE, 1 ml of stock LiAc), and vortex for ~30 sec. Incubate at 30 °C for 30 min with shaking at 200 rpm.
5. Add 80 µl of dimethyl sulfoxide (10% v/v final concentration) and mix well by gentle inversion. Heat shock for 15 min in a 42 °C water bath while mixing every 2 - 3 min.
6. Chill cell suspension on ice for 2 min and centrifuge at 14,000 x g for 15 sec at RT to recover yeast.
7. Resuspend cell pellet in 100 µl of 1x TE and plate on appropriate minimal SD medium plates for selective growth (medium lacking leucine and tryptophan to keep selective pressure on both bait and target plasmids).
8. Incubate plates upside-down at 30 °C until colonies are ~2 mm in diameter (usually 4 - 5 days). Plates can be stored at 4 °C for 2 - 3 weeks; for longer storage make glycerol stocks and store at -80 °C.
   NOTE: Verify that bait and target proteins are expressed in yeast by immunoblotting and that they do not have autonomous reporter gene activation when separately expressed in yeast.
9. Add 100 µl of the freshly prepared, competent yeast suspension and 600 µl of 1x LiAc/PEG solution (8 ml of stock PEG 3350, 1 ml of stock TE, 1 ml of stock LiAc), and vortex for ~30 sec. Incubate at 30 °C for 30 min with shaking at 200 rpm.
10. Add 80 µl of dimethyl sulfoxide (10% v/v final concentration) and mix well by gentle inversion. Heat shock for 15 min in a 42 °C water bath while mixing every 2 - 3 min.
11. Chill cell suspension on ice for 2 min and centrifuge at 14,000 x g for 15 sec at RT to recover yeast.
12. Resuspend cell pellet in 100 µl of 1x TE and plate on appropriate minimal SD medium plates for selective growth (medium lacking leucine and tryptophan to keep selective pressure on both bait and target plasmids).
13. Incubate plates upside-down at 30 °C until colonies are ~2 mm in diameter (usually 4 - 5 days). Plates can be stored at 4 °C for 2 - 3 weeks; for longer storage make glycerol stocks and store at -80 °C.
    NOTE: Verify that bait and target proteins are expressed in yeast by immunoblotting and that they do not have autonomous reporter gene activation when separately expressed in yeast.

2. Colony-lift Filter Paper β-Gal Assay

1. Prepare the following buffers:
   1. Prepare a Z-buffer containing 100 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM    KCl, and 1 mM MgSO₄; adjust the pH to 7.4. Sterilize by autoclaving and store at RT.
   2. Prepare X-Gal buffer by dissolving 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside at 20 mg/ml in N,N-dimethylformamide, and store in the dark at -20 °C. Prepare Z buffer/X-Gal solution. Make buffer prior to use by mixing X-Gal at 0.33 mg/ml and β-mercaptoethanol at 0.27% v/v in the Z-buffer. Use 2.5 ml per sample.
2. Add 2.5 ml of freshly prepared Z buffer/X-Gal solution to a clean 100 mm plate and place it inside a cellulose filter paper.
3. Place a new filter paper over the surface of the plate with the yeast colonies to be assayed. Gently rub the filter paper onto the plate with forceps and leave for ~5 min for colonies to attach.
   NOTE: Process the positive control in parallel, i.e., yeast co-transformed with pVA3 and pTD1.
4. Lift the filter paper and submerge it (with forceps) into a pool of liquid nitrogen for 30 sec (liquid nitrogen should be handled with care; always wear thick gloves and goggles). Let the frozen filter paper thaw at RT for ~2 min.
5. Place the filter paper (colony side up) on top of the pre-soaked filter paper inside the 100 mm plate, and incubate at 30 °C.
6. Check periodically (every ~30 min) for the appearance of blue colonies.
   NOTE: Weak bait: target interactions may take several hours to produce a positive blue signal (avoid prolonged incubation (>8 hr) that may give false positive results). For best results, use freshly co-transformed colonies, i.e., grown at 30 °C for 4 - 5 days, ~2 mm in diameter.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Plate incubator	Hereaus	B6120	Device
Orbital shaker incubator	New Brunswick Scientific	INNOVA 4300	Device
Spectrophotometer	Perkin Elmer	Lambda Bio+	Device
Matchmaker Two-Hybrid System 2 Kit	Takara Clontech	K1604-1	Contains bait vector pGBKT7, prey vector pACT2 and yeast strain Y190
Yeast Nitrogen Base	Sigma-Aldrich	Y0626	To prepare minimal SD growh medium
Dropout supplement lacking leucine and tryptophan	Sigma-Aldrich	Y0750	To prepare minimal SD growh medium
Dropout supplement lacking leucine, tryptophan, histidine	Sigma-Aldrich	Y2001	To prepare minimal SD growh medium
Herring testes carrier DNA	Takara Clontech	630440	For yeast transformation
Whatman filter paper Grade 5	Sigma-Aldrich	WHA1005070	For use in colony-lift filter paper β-Gal assay
X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)	Sigma-Aldrich	B4252	For use in colony-lift filter paper β-Gal assay