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19 Feb 2018

You are a researcher who has just discovered a new protein in a fungus. To help determine the protein's function, you want to identify the other proteins in the fungal cell with which your protein interacts. How do you design a yeast two-hybrid experiment to address this problem?

a) Express the protein in bacterial strain 1 as a fusion protein with the DNA-binding domain of Gal4p. Using bacterial strain 2, make a library representing all fungal proteins expressed as fusion proteins with the Gal4p activation domain. Encourage conjugation between strain 1 and strain 2, and look for colonies that express a reporter gene. These colonies will generally arise from cells containing fusion protein that interacts with your target protein.
b) Express the protein in yeast strain 1 as a fusion protein with the ribosomal-binding domain of Gal4p. Using yeast strain 2, make a library representing all fungal proteins expressed as fusion proteins with the Gal4p activation domain. Mate strain 1 with the strain 2 library, and look for colonies that express a reporter gene. These colonies will generally arise from mated cells containing fusion protein that interacts with your target protein.
c) Express the protein in yeast strain 1 as a fusion protein with the DNA-binding domain of Gal4p. Using yeast strain 2, make a library representing all fungal proteins expressed as fusion proteins with the Gal4p activation domain. Mate strain 1 with the strain 2 library, and look for colonies that express a reporter gene. These colonies will generally arise from mated cells containing fusion protein that interacts with your target protein.
d) Express the protein in yeast strain 1 as a fusion protein with the DNA-binding domain of Gal4p. Using yeast strain 2, make a library representing all fungal proteins expressed as fusion proteins with the Gal4p activation domain. Mate strain 1 with the strain 2 library, and look for colonies that express the target gene, which creates either a color or another selective advantage. These colonies will generally arise from mated cells containing fusion protein that interacts with your target protein.

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Hubert Koch
Hubert KochLv2
19 Feb 2018

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