Method to reduce false negative rate of yeast two-hybird assay

• 1、Department of Physiology and Pathophysiology, School of Basic Medicine Peking Union Medical College, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences 100005

Abstract： Objective: Protein-protein interaction is essential in multiple biological processes. Yeast two-hybrid is a classic method to study protein-protein interaction. LacZ is a commonly used reporter gene in this assay, which we found may increase the false negative rate of this method. Methods: Yeast two-hybrid assays were carried out to screen a random C-terminal peptide library with ZO-1 PDZ1 domain and GOPC PDZ1 domain as baits. The growth date and the degree of blue of the β-galactosidase activity tests were recorded. Plasmids from the positive clone and non-positive clone were both sequenced to analyze their sequence characteristics. Results: The degree of blue of LacZ assay is associated with the intensity of the interaction. The non-positive clones have similar sequence characteristics with the positive ones but with different affinity. Conclusion: The results indicate that the mainstream LacZ reporter gene produces many false negatives, which suggests that sequencing more clones may help to find more novel, weak, transient interactions.

Keywords： biological science yeast two-hybrid LacZ reporter gene false negative false positive weak PPI