Mapping Protein–Protein Interactions Using Yeast Two-Hybrid Assays

Transforming Yeast

• This method is suitable for transformation of bait or prey plasmid clones into their respective mating-competent, haploid yeast strains: Prey vectors are transformed into Y187, mating type “α,” while bait vectors are transformed into AH109, mating type “a.” The following is optimized for 100 transformations; a success rate of 100% per transformation should be expected. It also can be scaled down or up as required for high-throughput transformation, and most steps can be automated. As with all high-throughput procedures, small discrepancies can have noticeable downstream consequences. Verify the sterility and pH of reagents and the phenotype of yeast strains before transformation.

• 1. Inoculate 10–20 mL of YEPAD liquid medium with the appropriate haploid yeast strain. Grow overnight at 30°C in a shaking incubator (∼200 rpm, depending on model). After 12–16 h, measure the absorbance (OD₆₀₀) of the culture.

  • The OD₆₀₀ should be >1.5.

• 2. Dilute the cells in 100 mL of fresh liquid YEPAD medium to an OD₆₀₀ of ∼0.2–0.3. Grow the cells for ∼3–4 h (depending on the generation time of the strain) at 30°C in a shaking incubator (∼200 rpm).

  • The OD₆₀₀ should be between 0.5 and 1.0 (at least one doubling), indicating that the cells are in log phase of growth.

• 3. Transfer the culture to two 50-mL tubes. Collect the cells by brief centrifugation (∼700g for up to 5 min at room temperature) and discard the supernatant.

• 4. Wash the cells once with 25–50 mL sterile dH₂O. Collect the cells by brief centrifugation (∼700g for up to 5 min at room temperature) and discard the supernatant.

• 5. Resuspend the cells in 1 mL of 0.1 m LiAc (prepared in TE) and incubate on ice for 15 min.

• 6. Transfer the suspension to 1.5-mL microcentrifuge tubes and briefly centrifuge (∼350g for 30–60 sec). Discard the supernatant. Resuspend the cells in 1 mL of 0.1 m LiAc (prepared in TE) and incubate them on ice for another 15–20 min.

• 7. Prepare a transformation mix for ∼100 transformation reactions as follows.

  • i. Add 250 µL of carrier DNA (10 mg/mL) to 10 mL of 40% PEG in a sterile 50-mL tube. Vortex for at least 30 sec.

  • ii. Add 1–2 mL of freshly prepared competent yeast cells from Step 6. Vortex for an additional 60 sec.

• 8. Transfer 100 µL of reaction mix to each well of a 96-well plate using a multichannel pipette or robotic liquid handler.

  • Depending on the number of samples, the final volume can be divided such that each volume does not exceed 120 µL per well.

  • For fewer samples, 1.5-mL tubes may be used.

• 9. Add at least 100 ng of the desired plasmid DNA construct to each well and mix well with the pipette tip. Include one negative control containing carrier DNA but no plasmid DNA and at least one positive control containing the empty vector construct.

• 10. Seal the 96-well plate with adhesive aluminum foil and Parafilm to secure the edges.

  • All exposed plate surfaces should be sealed to protect against contamination.

• 11. Vortex the plate gently for 3–4 min, ensuring that the wells do not cross-contaminate.

• 12. Place the plate for 45 min at 30°C with gentle shaking (∼60 rpm).

• 13. Transfer the plate to a water bath at 42°C and incubate for 30 min. Ensure the plate is floating and water is not entering the wells.

  • For small scale transformation using microcentrifuge tubes, this incubation may be reduced to 15 min.

• 14. After 30 min, centrifuge the plate at ∼700g for 8–10 min at room temperature. Discard the supernatant by multichannel pipette and rinse the plate surface with 95% ethanol to avoid cross-contamination between wells.

• 15. Resuspend the contents of each well in 50–100 µL of sterile dH₂O. Spread each volume on a separate Omnitray plate (or distinct sections of a Qtray) containing selective solid medium.

• 16. Incubate the plates for at least 2–3 d at 30°C.

  • Some strains, such as Y187, may require 3–4 d to yield noticeable transformant growth.

  • Plates may be stored for several months at 4°C as long as the medium does not dry out or become contaminated. For long-term storage, transformants may be cultured in rich (YEPAD) or selective liquid medium overnight, resuspended in 20% glycerol, and transferred to a freezer at −80°C.

• 17. After preparation of the desired bait transformants, proceed to self-activation testing of baits (Steps 18–23) followed by library-based (Steps 24–35) and/or array-based (Steps 36–44) screening.

Self-Activation Testing of Bait Transformants Using 3-AT

• Performing self-activation testing before Y2H screening helps optimize screen conditions, saving time and resources. 3-AT is a competitive inhibitor of the HIS3 gene product and is frequently used in Y2H screening to increase the stringency of selection when using the HIS3 reporter gene. The following assay measures the background activity of the HIS3 reporter in baits in the presence of empty prey vector.

• 18. Prepare 5-mL cultures in YEPAD liquid medium using empty-vector transformants in the prey strain (i.e., the Y187 strain containing the pGADCg empty vector). Grow the cultures for 12–16 h at 30°C in a shaking incubator (∼200 rpm).

• 19. Plate the bait clones on YEPAD using a sterilized plate replicator.

  • Either the standard 96-spot or 384-spot format can be used, depending on the number of clones.

• 20. Perform yeast mating between the bait clones and the prey strain carrying empty vector as follows.

  • i. Transfer the empty-vector prey culture from Step 18 to an empty Omnitray plate.

  • ii. Touch the liquid culture with the pins of a sterilized plate replicator, then touch the pins directly onto the plated bait spots from Step 19.

  • iii. Incubate the cells for 36–48 h at 30°C to allow mating.

• 21. To select for diploid colonies, transfer the colonies to plates containing the appropriate double-dropout selective medium (–LT) using a sterilized plate replicator. Grow the cells for 2–3 d at 30°C until the colonies are 1 mm in diameter.

• 22. To measure HIS3 reporter gene activity, transfer the diploid colonies to plates containing the appropriate triple-dropout selective medium (–LTH) plus 3-AT at the following concentrations: 0, 1, and 3 mm.

  • The initial 3-AT concentrations may be increased to 10, 25, 50, or 100 mm if necessary. Higher 3-AT concentrations are inadvisable as they will halt all growth.

• 23. Incubate the selective plates for ∼1 wk at 30°C. Record the lowest 3-AT concentration at which background colony growth is completely prevented.

  • The recorded concentration of 3-AT should be added to selective plates in the subsequent interaction screens to suppress reporter activation in the absence of an interacting prey.

Screening for Protein Interactions Using Genomic/cDNA Libraries/ORFeomes

• In random library screening (Fig. 2), a single bait of interest is screened against a full library of prey proteins in a single screen. The interacting prey partner is identified by sequencing.

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Figure 2.

Y2H random library screen. The bait construct (DBD fusion protein) and the prey library (AD fusions with a cDNA or random genomic library) in haploid yeast strains are mixed in a 1:1 ratio. The mating culture is plated on a YEPAD agar plate. After incubation of mating plates for 6 h at 30°C, the diploid cells are transferred to –LTH plates (with or without 3-AT) for selection. The interacting preys are then identified by PCR amplification and sequencing of the vector inserts.

Screening for Protein Interactions Using Arrays

• After baits are screened for potential self-activation (Steps 18–23), they may be tested for interactions versus the prey array as follows. Arrays are used as a primary screen or to validate the positives resulting from a library screen. Given the built-in controls present in array-based screens, they are the best choice for differentiating strong and weak interactions from background and efficiently reducing false positives. Usually, preys rather than baits are arrayed in a defined order, as described here, due to the high self-activation potential among baits; the activation domain (AD) used with prey proteins does not generally produce self-activation. An overview of array-based screening is provided in Figure 3.

• Whether screening is manual or automated, a high-density (96-pin or 384-pin) replicating tool is used for routine transfer of colonies from one plate to another. In the procedure described here, the prey array is gridded on single-well (86 × 128-mm) Omnitray plates in a 96- or 384-colony format.

• 36. After preparation of the desired prey transformants (each carrying one specific AD-containing plasmid), array the preys on plates containing selective solid medium (–L) in either a 96- or 384-format in duplicates or quadruplicates.

  • Baits can be arrayed likewise, as needed. Other array formats such as 768 or 1536 colonies per plate can be used, but the higher densities may render scoring positives more difficult.

  • For long-term storage, all arrays may be cultured in rich (YEPAD) or selective liquid medium overnight, resuspended in 20% glycerol, and transferred to a freezer at −80°C. For day-to-day use, the arrays can be stored on solid selective medium (−L or −T) for up to 3 mo at 4°C. Fresh array plates may be produced using these storage plates before each screen. Copying the array onto fresh selective plates every 2 wk should protect against plasmid loss.

• 37. Use the sterile plate replicator to transfer the yeast prey array to plates containing YEPAD solid medium. Grow the array overnight at 30°C.

  • Alternatively, a fresh prey array can be prepared in YEPAD liquid medium in a 96-well plate. Note that only the master prey array (Step 36) should be used to make fresh copies for mating.

• 38. Transfer 15–20 mL of liquid YEPAD medium to a 50-mL tube. Inoculate the medium with the bait strain (or pool of baits) and grow overnight at 30°C in a shaking incubator (at ∼200 rpm).

  • A bait pooling strategy may be used to reduce the time of Y2H screening. The pool of baits may be grown together in 15–20 ml YEPAD broth overnight.

  • If the bait strains are frozen, they should be streaked or pinned on selective solid medium plates and grown for 1–2 d at 30°C before any experiment. A fresh bait culture should always be prepared for Y2H mating, as extended culturing in rich media may result in plasmid loss.

• 39. Dip the sterile plate replicator in the bait liquid culture and transfer the cells onto a plate containing YEPAD solid medium. Replicate as needed. Allow the spots to dry for 15–20 min.

  • Ensure YEPAD plates are free of residual moisture before pinning to ensure consistent colony formation.

• 40. Touch the fresh prey array cultures from Step 37 with the sterilized plate replicator and transfer them directly onto the plated bait spots. Ensure that each of the bait spots receives different prey cells (i.e., a different AD-fusion protein).

• 41. Incubate the cells for 36–48 h at 30°C to allow mating.

  • Supplemental adenine in the bait culture medium may increase the mating efficiency of some baits. Optimization of this step is subject to yeast strains, bait and prey vectors, and laboratory conditions.

• 42. To select diploid cells, transfer the colonies from the mating plates to plates containing double-dropout selective solid medium (–LT) using the sterilized plate replicator. Grow the cells for 2–3 d at 30°C until the colonies are 1 mm in diameter.

  • This step ensures successful mating, as only diploid cells containing the selection markers in the prey and bait vectors will grow. Low mating efficiency will noticeably reduce the quality of screen results by obscuring signal and noise.

• 43. Transfer the colonies from –LT plates to plates containing triple-dropout selective solid medium (–LTH, with or without 3-AT) using the sterilized plate replicator. Incubate the plates for 6–8 d at 30°C.

  • The choice of 3-AT concentrations will depend on the results of the bait self-activation test (Steps 18–23).

• 44. Examine the plates and score the positive interactions by counting colonies that are significantly large in size compared with background. If colonies have been plated in duplicate or quadruplicate, at least half of the colonies in each set should be significantly larger than background growth to indicate a positive result.

  • Plates should be examined and monitored every day for positive colonies and contamination. Most Y2H positive colonies should appear within 3–5 d but some positive interactions may require more time. For most screens, observing >10 positive results per set of 96 different preys constitutes a suspicious result and may indicate false positives.

  • See Troubleshooting.

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Figure 3.

Array-based two-hybrid assays. Bait (pGBKCg, pGBGT7g) and prey (pGADCg, pGADT7g) constructs are transformed into haploid yeast cells of opposite mating type (a and α). Preys are arrayed on selective solid medium (–L). Baits should be tested for self-activation. The Y2H screening begins with mating, in array format, of two haploid strains carrying bait and prey plasmids. Diploid cells are selected by transferring the mated array to double-dropout (–LT) followed by triple-dropout (–LTH) plates. The rectangles on the selective plate (bottom panel) mark negative and positive interactions between the bait and the prey at these specific positions of the array.