Encapsulin Nanoparticle Engineering for Enhanced Cofactor Regeneration

• Valerie Arreola, Biological Sciences, Columbia University

• Wilfred Chen, Biomolecular and Chemical Engineering, University of Delaware

Encapsulins are a kind of nanoparticle, originating from prokaryotic agents, that have a wide variety of applications. The attractiveness of the nanoparticles originates from their capacity to encapsulate large protein cargo whilst also being able to be externally decorated by various agents. This project aims to explore such qualities of external decoration through various  biomolecular engineering approaches as well as explore previously engineered enzyme pathways for enhanced cofactor regeneration through the nanoparticle scaffold. However, in exploring such pathways for cofactor regeneration enhancement, it must first be assessed that the insertion sites in which the tags utilized to create the enzyme scaffold, SpyTag and DogTag, do not affect the solubility & assembly of the nanoparticle itself or reactivity of each component in the scaffold. Each of the qualities affects how encapsulin nanoparticles will function in a foreign environment and must be analyzed before proceeding with exploring enzyme cofactor regeneration. Solubility and assembly of the nanoparticles were assessed through SDS-Page analysis and ELP purification, respectively. The data gathered from such analyses was able to provide the information needed to determine which insertion variants are most suitable for the implementation of an enzyme pathway that enhances cofactor regeneration. The scaffold will then be subjected to a PK/LDH analysis to determine if cofactor regeneration is enhanced. Overall, this project aims to provide an accurate prediction of how encapsulin nanoparticles can be explored for various applications, including carbon capture efforts.