Reprogramming Yeast Mating to Characterize Protein-Protein Interactions in High-Throughput

Advances in computational protein design allow for the rapid generation of predicted synthetic protein-protein interactions (PPIs), which are used in downstream applications ranging from medical therapeutics to synthetic biology. Despite improvements with in silico modeling, experimental screening is necessary for identifying and optimizing designs that meet specified criteria and is a major bottleneck for PPI engineering. We have developed yeast synthetic agglutination (YSA) to achieve quantitative library-on-library characterization of PPIs in a controllable environment by reprogramming the sexual agglutination of S. cerevisiae to link protein interaction strength with mating efficiency. Validation of YSA with 96 previously characterized interactions shows a strong log-linear relationship between mating efficiency and PPI strength for interactions with a K_D between 25μM and 100pM. Using induced chromosomal translocation to pair barcodes representing interacting proteins, up to at least 7,000 distinct interactions can be screened in a single pot. To demonstrate the screening of dynamic interactions that respond to environmental changes, we show that the addition of non-membrane soluble competitor to the binding environment generates a PPI disruption profile that perfectly matches our expectation. YSA enables the high throughput quantitative characterization of engineered PPIs in an extracellular environment and removes the experimental bottleneck for many new and exciting protein engineering challenges.