Studies on the self-assembly of geometrically designed protein fusions using genetically programmed chemistry

Phd thesisSelf-assembling proteins have great potential to be applied to a myriad of biotechnological applications. The engineering of these proteins to create modules that will assemble into user-defined structures is a key challenge in their development. Several bespoke solutions exist, however the creation of a generic multi-purpose approach is a complex task. This thesis investigates the creation of an expandable recombinant protein assembly system that produces scalable superstructures via split intein mediated native chemical ligation. Using repeat protein “building blocks” we will demonstrate the design of controllable fibre formation and scalable protein cages. To create a sequentially produced fibre two types of construct are used: a double-sided building block and a one-sided capping protein. Scalable protein cages are formed using oligomeric multimers as vertices to create two mirror-image “half cages”, these can be expanded through the addition of a double-sided building block. To demonstrate the versatility of this methodology modules were ligated in both one-pot and stepwise reactions. Using mild conditions rapid formation of protein superstructures was observed and discrete fibres and nanocages were purifiable.Leverhulme Trust (Grant no: RPG-2014-089