Recombinant Expression and Purification of High Aspect Ratio Proteins with Terminal Bioconjugation Handles

• Craig Roberts, Chemical Engineering, University of Delaware

• Antonio Goncalves, Chemical & Biomolecular Engineering, University of Delaware

Helical bundles, structural motifs commonly found in proteins, consist of multiple alpha helices packed together in a specific arrangement. These bundles play crucial roles, including providing structural support and facilitating protein interactions. In this research project, helical bundles are employed to separate two catcher binding sites, facilitating the formation of conditional cellular outcomes in conjunction with Cage and Key proteins, which mediate the cellular response. This structure also serves as a backbone for fluorescent signaling and can be extended into strings using alternating Dog Tag/Catcher and Spy Tag/Catcher pairs, enhancing structural integrity and reducing degradation during physiological delivery compared to a single bundle of the same length. Cancer cells, often characterized by the overexpression of specific receptors that facilitate uncontrolled replication, can be targeted for treatment by exploiting this attribute. The designed protein strings will preferentially bind to cancer cells with higher receptor densities, effectively delivering therapeutic signals to these cells. Utilizing plasmid biochemistry, these proteins benefit from a rapid design-to-expression timeline, with quick cloning and protein formation that can be tested promptly. Post-expression, the lysate is purified using designed purification tags to extract the desired protein for implementation, improving yield compared to other purification methods.