UC Berkeley

The ability to construct site-specific protein bioconjugates has a range of applications in basic and applied science. Many biorthogonal chemistries have been developed to modify proteins at well-defined sites, but these often require the introduction of artificial functionality into a protein sequence, a laborious process that comes at the cost of protein expression. An alternative approach targets native functionality present in a protein sequence, such as the N- or C-terminus, for which most proteins have one. Herein I developed a suite of enzymatic methods for the modification of protein termini all using the enzyme tyrosinase. In an initial demonstration I showed that tyrosinase can oxidize small molecule phenols, generating a reactive o-quinone intermediate, which covalently modifies protein N-termini. This strategy has a number of key advantages, including the use of catalytic amounts of commercially available enzyme and easily accessible reagents. This work laid the foundation for a new research area in the lab focused on the development of tyrosinase-mediated bioconjugations. I then worked in a collaborative team effort to extend this approach to protein C-termini, where tyrosinase could be used to generate site-specific o-quinones at introduced tyrosine-tags. These could couple to free thiols on a different protein, allowing for the rapid and facile synthesis of protein-protein conjugates. I ultimately applied this method towards the modification of the cell surface, where I used tyrosinase to synthesize nanobody-natural killer conjugates that could elicit targeted cell killing. Together, these methods have enabled the synthesis of complex bioconjugates that were previously difficult to access.