Design of Modular Polyhydroxyalkanoate Scaffolds for Protein Immobilization by Directed Ligation.

In vivo-assembled polyhydroxyalkanoate (PHA) particles have been successfully bioengineered to display foreign protein functions toward high-value applications in medicine and industry. To further expand the design space of PHA particles toward immobilization of various functional proteins, we developed a tunable modular protein immobilization method implementing the SpyCatcher/SpyTag chemistry. We successfully displayed the SpyCatcher protein using translational fusion with the Ralstonia eutropha PHA synthase (PhaC). The SpyCatcher domain displayed on the surface of PHA particles was accessible for cross-linker-free ligation with SpyTag-bearing proteins. We demonstrated tunable protein immobilization of various SpyTagged proteins on SpyCatcher-PHA particles, which ultimately enabled assembly of multiple proteins coating the surface of PHA particles. Overall, the functionality, stability, and reusability of proteins immobilized to SpyCatcher-PHA particles were either retained or enhanced in comparison to the soluble forms. This modular platform can be implemented as a generic tool for protein immobilization in an array of applications.