This article describes the yeast two-hybrid assay, a method used to study protein-protein interactions. The process involves genetically engineered yeast cells that can synthesize leucine and tryptophan when two hybrid proteins interact.
For the yeast two-hybrid assay, take a genetically engineered, actively-growing yeast cell suspension lacking the transcription activator protein, GAL4. These cells cannot synthesize leucine and tryptophan.
Add a pair of expression plasmids: one encoding leucine and the DNA-binding domain, DNA-BD, of GAL4, fused to a target protein domain, and the other encoding tryptophan and the activator domain, AD, of GAL4 fused to another domain of the target protein.
Add a buffer containing suitable agents for transformation. Heat-shock the cells to cause transient pore formation, facilitating cellular plasmid uptake. Cool to reseal the pores. Plate the cells on a selective growth medium lacking leucine and tryptophan.
The transformed cells expressing two hybrid proteins synthesize leucine and tryptophan, survive, and form colonies.
The DNA-BD fused to a protein domain binds to the specific upstream DNA sequence of the β-galactosidase-encoding gene promoter region. If the target protein domains interact, the AD and DNA-BD come closer, causing the reconstitution of functional GAL4, leading to expression of β-galactosidase gene.
Transfer the colonies onto filter paper. Freeze-thaw the cells to lyse them and release β-galactosidase. Place this filter paper over another filter paper soaked with a β-galactosidase substrate.
The released β-galactosidase hydrolyzes the substrate to galactose and an unstable product, which dimerizes and gets oxidized forming a blue precipitate on the filter paper, indicating target protein domain interaction.
In this yeast two-hybrid assay, yeast cells in mid-log phase are used for transformation. Harvest the cells by centrifugation. Resuspend each pellet in five milliliters of sterile water, and pool the cell suspensions. Centrifuge the cell suspension.
After discarding the supernatant, resuspend the yeast pellet in one milliliter of freshly-prepared sterile 1X lithium acetate and TE. The competent yeast cells must be used within one hour of preparation. Prepare plasmid samples by mixing bait and target plasmid DNA with 100 micrograms of Herring testes carrier DNA.
To each 1.5-milliliter tube, add 100 microliters of the competent yeast suspension and 600 microliters of 1X lithium acetate and PEG solution, and vortex for about 30 seconds. Incubate at 30 degrees Celsius for 30 minutes with shaking. Add 80 microliters of dimethyl sulfoxide, and mix well by gentle inversion.
Heat shock for 15 minutes in a 42-degree Celsius water bath while mixing every two to three minutes. Chill the cell suspension on ice for two minutes, and then, centrifuge to recover the yeast. Resuspend each cell pellet in 100 microliters of 1X TE. Plate the cells on appropriate minimal SD medium plates to keep selective pressure on both bait and target plasmids. Incubate the plates upside down at 30 degrees Celsius for four to five days.
The yeast colonies are ready for this assay when they are about two millimeters in diameter. Pipette 2.5 milliliters of freshly-prepared Z-Buffer and X-Gal solution to a clean 100-millimeter plate and place a cellulose filter paper in the plate. Place a new filter paper over the surface of the plate with the yeast colonies to be assayed. Use forceps to gently rub the filter paper onto the plate and leave for approximately five minutes for the colonies to attach.
While using appropriate personal protective equipment, carefully lift the filter paper and submerge it into a pool of liquid nitrogen for 30 seconds. Let the frozen filter paper thaw at room temperature for two minutes. Place the filter paper with the colony side up on top of the pre-soaked filter paper inside the 100-millimeter plate and incubate at 30 degrees Celsius until blue colonies appear.
繁體中文
