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