Protein-based constructs are valuable moieties for a variety of biological and therapeutic applications. Enzymatic protein modification coupled with subsequent bioorthogonal reactions has enormous potential for the creation of a diverse range of site-specifically modified conjugates. Protein prenyltransferases catalyze the covalent modification of proteins that possess a C-terminal tetrapeptide CaaX-box sequence; virtually any protein containing such a tag is a substrate. Here, three applications of this enzymatic labeling approach will be described. In the first, prenylation with natural isoprenoids of fibronectin domain-based dihydrofolate reductase (DHFR) fusion proteins and subsequent assembly into chemically self-assembled nanorings allowed lymphocytes to be targeted to cancer cells for directed killing. Next, an aldehyde-containing isoprenoid analogue was transferred to a DARPin (Designed Ankyrin Repeat Protein) scaffold engineered to target cancer cells. Oxime ligation using a TAMRA-based reagent allowed visualization of cancer cells while a different MMAE-based (monomethyl auristatin) reagent with that aldehyde-containing construct produced a protein that was able to selectively kill cancer cells. In a third application, a polymer initiator was enzymatically installed onto the above DARPin protein followed by polymerization with monomers containing bioorthogonal azides and alkynes to yield protein-polymer conjugates with improved pharmacological properties. Subsequent elaboration of the azides and alkynes with fluorphores and toxins enabled simultaneous visualization and targeting of these proteins to cancer cells. These three examples highlight the enormous versatility of this approach.

Enzymatic protein modification enables the creation of multifunctional macromolecular assemblies for therapeutic applications

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.