Controlling the loading of protein nanocages

The work described in this thesis focuses on the use of natural protein building blocks to assemble nanocages. Most common strategies for the loading of different protein nanocages suffer from a lack of numerical control over cargo loading. The aim of this thesis was to create a controllable methodology to selectively load protein nanocages with the cargo of interest. It focuses on two fundamental approaches to optimize the loading: modifying the cargo by genetically engineering and exploring different protein cages. The capsid of the Cowpea Chlorotic Mottle Virus (CCMV) was used as a well-established platform for the design of new cargo loading techniques. Loading could be controlled up to ~20 fluorescent proteins per capsid, however, higher cargo loading ratios led to erroneously formed capsids. A new type of protein nanocage was also explored: the bacterial encapsulins. Overall, the encapsulation of non–natural cargo in encapsulins is highly effective, yet still a very novel approach in which many things remain to be investigated. From this thesis, it is apparent that the best control over protein nanocage loading is achieved in vitro, by purifying the cargo and shell proteins and carefully calculating the mixing ratios.