High-Throughput Protein-Protein Interaction Screening Using Cell-Free Protein Synthesis


Protein-protein interactions are ubiquitous in living systems, and mediate important cellular processes from decision making to immunity against pathogens. Furthermore, protein-protein interactions are key to many protein therapeutics, pathogen diagnostics, and numerous synthetic biology applications. As a result, there has been significant effort to develop methods to express potential protein interaction partners and evaluate their protein-protein interactions in high-throughput. However, despite decades of development, many methods are still bottlenecked by labor intensive and poorly scalable steps involving cell culture. In this dissertation, I describe my efforts to build a high-throughput protein expression platform leveraging cell-free protein synthesis (CFPS) and acoustic liquid handling robotics and my efforts to apply this method to the study and engineering of protein-protein protein interactions. Towards this goal, I first developed methods to express and screen computationally designed protein heterodimers. In collaboration with researchers in the Baker lab, we sought to use these heterodimers to design protein-protein interaction-based logic gates to mediate post-translational control of biological systems. Leveraging CFPS and a nanoluciferase-based complementation reporter, I evaluated the pairwise interactions of computationally designed heterodimers and used their interaction map to construct induced dimerization, AND, OR, and NOR logic gates. I also showed that the gates can rapidly integrate information, with the induced dimerization gate exhibiting a 7-fold induction within 5 minutes of activation. In collaboration with others, we also found that protein-protein interaction-based logic gates also operate in both yeast and human T cells, highlighting this promising strategy for post-translational control of biological behavior. I next turned my efforts to develop high throughput methods for the discovery of antibodies. I showed that a crude extract based CFPS system can support the expression and assembly of functional antibody fragments from linear DNA templates in 2 μL CFPS reactions in 384-well plates. Using the AlphaLISA high-throughput protein-protein interaction assay, I analyzed the interactions of antibodies and their antigens in crude CFPS reactions without purification. To highlight the capabilities of the platform, in less than 24 hours I expressed and screened 120 previously reported neutralizing antibodies targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein. The developed method would have enabled the discovery of 10 out of the 13 most potent antibodies screened, is end-to-end automatable, and has more than 10x the throughput and is more than 3.5x faster than state-of-the-art antibody discovery workflows. I next led an effort to develop computationally designed multivalent minibinders that inhibit the key protein-protein interaction that enables SARS-CoV-2 to enter host cells. Leveraging similar methods to those developed for the antibody screening workflow, I expressed and screened hundreds of multivalent minibinders and identified candidates that bound with high affinity to the SARS-CoV-2 spike glycoprotein of the original Wuhan-Hu-1 variant and all other tested variants of concern. The top design, TRI2-2, exhibits an apparent dissociation rate slower than 10-7 s-1, simultaneously engages all three receptor binding domains of the spike glycoprotein, and potently neutralizes all tested SARS-CoV-2 variants including Delta (B.1.617.2) and Omicron (B.1.1.529 or BA.1). Furthermore, TRI2-2 confers protection against SARS-CoV-2 when administered intranasally in mice, indicating that TRI2-2 is a promising potential therapeutic for the treatment of the coronavirus disease 2019 (COVID-19). In a subsequent collaborative effort, I adapted the TRI2-2 multivalent minibinder for the detection of SARS-CoV-2 infection in a nanomechanical biosensor. The developed sensors enable detection of the SARS-CoV-2 spike glycoprotein antigen in less than 5 minutes with a limit of detection more than two orders of magnitude better than state-of-the-art lateral flow antigen tests and on par with that of state-of-the-art nucleic acid amplifications tests like the quantitative reverse transcription polymerase chain reaction (RT-qPCR). Taken as a whole, the work in this dissertation provides advancements in the high-throughput expression proteins and the analysis of protein-protein interactions.

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