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

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

Blog Article

The yeast reverse two-hybrid system is a powerful technique for isolating mutations in a protein that abolish its interaction with a known partner.Selection is based on abrogation Nesting Chairs 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 protein (GFP) to the C-terminus of Methylcobalamin a protein of interest, dynein light chain (LC8), we were able to rapidly isolate mutations that did not result in protein truncation.