Rapid Selection against Truncation Mutants in Yeast Reverse Two-Hybrid Screens

Rapid Selection against Truncation Mutants in Yeast Reverse Two-Hybrid Screens

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The yeast reverse two-hybrid system is a powerful technique for isolating mutations in a Fan protein that abolish its interaction with a known partner.Selection is based on abrogation of growth suppression imposed when wild-type interactions confer 5-fluoroorotic acid (5-FOA) sensitivity to yeast cells.A laborious component of this system is to eliminate those mutations that cause protein truncation.

By fusing the green fluorescent Environmental protein (GFP) to the C-terminus of a protein of interest, dynein light chain (LC8), we were able to rapidly isolate mutations that did not result in protein truncation.