Natural proteins often assemble into various complex geometric structures based on their interactions with each other. The King Lab at the University of Washington's Institute for Protein Design uses the way these proteins behave to develop computational models that enable the design of novel self-assembling protein cages, or nanoparticles. The designed particles are capable of holding and transporting molecules or displaying antigens on their surface, making them effective vaccine candidates. My project involves recovering the solubility of one of these protein cages known as T33_dn2. T33_dn2 is a tetrahedral protein cage comprised of four copies each of two trimeric components known as T33_dn2A and T33_dn2B. While both components can be expressed individually through E.coli before being assembled in vitro, they can also be expressed bicistronically and assemble in vivo. Currently, the use of T33_dn2 as a vaccine scaffold is limited because T33_dn2B is insoluble, and only seems to be stabilized in solution when associating with T33_dn2A. When expressed bicistronically, however, the cage has an extremely low yield. For a protein to be developed into a vaccine, it must be soluble. To recover the solubility and yield of T33_dn2B, I am testing ten plasmid variants of bicistronic T33_dn2. The “original” plasmid consists of one gene coding for a high-expressing cleavable SUMO protein attached to T33_dn2A and another coding for T33_dn2B. The additional nine variants have single point mutations at specific locations on the T33_dn2A gene intended to affect binding strength. After expression, introducing wildtype T33_dn2A in vitro will allow for the formation of T33_dn2. I will be presenting the results of these expression, purification, and assembly tests.