UCLA

Biological containers known as protein cages are widespread in nature—displaying unique functionality via their ability to compartmentalize molecules in an internal lumen volume. Composed of repeated copies of a small number of protein subunits arranged symmetrically, protein cages are relatively simple in composition, and yet complex in structure. In nature these assemblies function in transport and protection, storage of important molecular cargo, and sequestration of metabolic reactions. These demonstrations of incredible functionality have inspired researchers seeking to engineer complex protein architectures with diverse functionality to direct their attention towards symmetric protein cages. Resulting from these efforts, a modest number of protein engineering studies have succeeded in replicating these structures de novo in hopes of applying them to the fields of medicine and biotechnology; however, developing applications for these assemblies that outperform existing technologies remains a challenging exercise. The primary aims of my thesis work are the design and development of applications for symmetric protein cages with a particular focus on mimicking natural functionalities not yet recreated via artificial protein design methods. We note the limitations of prevalent protein design methods, and technological advances that may revolutionize the field.The first chapter of this thesis is a brief introduction to protein cages as platforms for engineering, as well as efforts to design them de novo in the laboratory. In the second chapter of this thesis we sought to advance on existing methods for protein cage engineering by designing new cages with novel interface construction. These cages incorporate a grafted interface onto each of two components making up the cages. In this work we describe challenges associated with direct rigid linkage of protein components via alpha helical extension and discuss whether the technique is a viable strategy for future cage design efforts. Next we attempt a novel approach to addressing a long-standing challenge in the field of protein design: engineering structurally responsive protein cages. The natural world holds numerous examples of protein cages that can assemble and disassemble in response to specific stimuli, but recreating this effect in the laboratory has proven challenging. In chapter three we present work on the design of protein cages that disassemble in response to a specifically chosen target protease. This work related to protein cage disassembly has implications in therapeutic design, as proteases are common therapeutic targets, and developing systems responsive to diseases biomarkers could be of great utility. In the next chapter we expand our focus on cage disassembly to other types of triggers for cage disassembly. In this work, we devise a system utilizing antibody-mimetic molecules called DARPins that allows for the design of protein cages that disassemble in response to any protein of interest. The work in this chapter focuses on a natural cage assembly (sulfur oxygenase) as a platform for development of this effect. The fifth chapter of this thesis covers work related to designing protein-cage based nanoparticles to be used as immunosorbents for patients on dialysis. The work addresses a clinical need for methods to remove the protein beta-2 microglobulin from the blood, as high concentrations are common in patients on long-term hemodialysis therapy, but can be toxic. We describe a protein cage that displays nanobodies on its exterior surface to generate beta-2 microglobulin adsorbent cages (BACs). In conclusion, the research performed in this dissertation work seeks to advance on existing methods for protein cage engineering by designing new cages with novel interface construction. I then used previously described artificial protein cages to demonstrate triggerable protein cages with dynamic assembly properties that are responsive to molecular stimuli, as well as developing of an immunosorbent nanoparticle with clinical applications.