Protein cages such as virus-like particles (VLPs) are nanoscale structures with significant potential in biotechnology and medicine, serving roles in drug delivery, imaging, and vaccine development. Enhancing their functions often relies on directed evolution, which depends on a strong genotype-phenotype linkage naturally present in VLPs that package their own genetic material. However, many DNA-free protein cages, including prokaryotic protein cages known as encapsulins, lack this linkage because they self-assemble without encapsulating their encoding DNA. This absence poses a challenge for applying directed evolution to these structures.
To overcome this limitation, we have engineered a novel gene circuit that creates a survival-based selection system linked to specific desired features of encapsulins. In our system, only cells expressing fully assembled cages with those desirable features survive, while those with unassembled or misassembled components are eliminated. This approach effectively establishes a genotype-phenotype linkage by tying cell survival—and thus the genetic material—to successful evolution.
In this presentation, I will detail the design of our gene circuit, the construction of encapsulin libraries, the directed evolution screening process, and the novel encapsulin variants we have evolved. Our work provides a solution to a key challenge in protein engineering and opens new avenues for the development of advanced biomaterials and nanodevices.