| Description |
Subcellular protein compartmentalization is one of the strategies that nature has adapted for various functions, such as storage and protection of cargo molecules as well as regulation of chemical reactions. There is a growing interest among nanotechnologists to utilize protein capsids for encapsulating guest molecules in bioimaging, drug-delivery, nanoreactors, etc. To date, many viral and nonviral protein capsids have been investigated for those applications. We used an engineered an Aquifex aeolicus lumazine synthase variant (AaLS-13) as a platform to encapsulate a guest enzyme by charge complementarity. The enzyme, esterase Est55 bearing a C-terminal deca-arginine (R10) tag, was placed in the lumen of the AaLS-13 during the self-assembly of the capsid when co-produced in E. coli. This encapsulated enzyme was characterized by colorimetric assay with the substrate, pnitrophenyl acetate. Comparison of the specific esterase activity between the empty capsid and the enzyme-loaded capsid showed 64-fold higher specific activity in the enzyme-loaded capsid compared to the empty capsid. Mass spectrometry and proteomics analysis verified that the presence of Est55-R10 in a sample of purified AaLS-13 that had been co-produced with the esterase. Capsid loading was estimated by esterase activity measurements and SDS-PAGE analysis. Both methods gave a loading estimate of one esterase molecule per eleven capsids. We plan to improve capsid loading using "supercharged" esterases. We also plan to investigate the porosity of the capsid because size limit for small molecule diffusion in and out of capsid is not well defined. Esterase substrates of various sizes will be applied to both esterase-loaded capsid and free esterase to determine the molecular weight cut-off of the AaLS-13 capsid. |
