A Cell-Free Protein Synthesis Platform for Rapid and Quantitative Protein-Protein Interaction Analysis

Protein-protein interactions (PPIs) facilitate many core biological functions, from signaling to complex and compartment assembly. Despite their importance and ubiquity, the study of PPIs is still often laborious and time-consuming. Additionally, there are few methods for the investigation of PPI kinetics in high throughput, despite the fact that understanding interaction kinetics is often critical for understanding and designing biological function. In this work, we present a robust cell-free method for the characterization of PPIs that enables high throughput, rapid, and quantitative characterization of both PPI interacti­on networks and kinetics. Our platform leverages cell-free protein synthesis, an Echo^® 525 acoustic liquid handler, and a split luciferase complementation reporter to enable rapid expression and characterization of tens to hundreds of interaction partners on short time scales. We will present our results to validate the platform and characterize novel PPIs. Specifically, we will validate the method by measuring the kinetics of the well-studied PUMA and MCL-1 model PPI system without purification. Additionally, we will show our efforts to validate the platform's ability to characterize novel PPIs by examining tens of novel de novo designed heterodimers. A key feature of our approach is the ability to measure both the (1) interaction network and (2) kinetics of all strongly interacting and stable heterodimers within a single platform. Our results show that cell-free protein synthesis coupled with liquid handling robotics is a powerful tool for the rapid and high throughput characterization of PPIs. We expect this method will accelerate both the study and engineering of PPIs.