E. coli Based Genetic Selection System: A Novel Toolkit for PROTAC and Drug Discovery

PROTAC (PROteolysis TAging Chimera) is an immerging approach that allows the elimination of undruggable proteins (such as cancer drivers). Contrary to RNAi and CRISPR gene editing techniques that blocks or remove the gene of interest, PROTAC is a small molecule drug that promotes the ubiquitylation and degradation of the gene product. These molecule degraders have two functional groups, one interacts with ubiquitin E3-ligase and the other with the protein of interest. Tagging the undruggable protein with ubiquitin promotes its degradation and therefore can function for cancer and other disease therapy. To identify new PROTACs pharma use mammalian cells based system with GFP-like fusion as reporters for degradation. Such screening systems are complicated, expensive and result in numerous false positive hits. Therefore, there is an unmet need for tools that will facilitate the discovery of new PROTACs.

Ubiquitylation is the major signal for protein degradation in eukaryotes but it lacks in prokaryotes. We take synthetic biology approach to functionally express the ubiquitylation apparatus in E. coli. Recently, we developed a novel E. coli based positive genetic selection system for ubiquitylation. We designed a novel split antibiotic resistance protein system that facilitate efficient studies of protein-protein or protein-drug interactions as well as protein ubiquitylation. We demonstrated the system response to thalidomide and lenalidomide, (FDA approved PROTACs), and generated new high throughput screening systems for various diseases known targets.

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