Nanoparticles are versatile particles ranging from 1 to 100 nm. They have potential applications in various fields, including biomedical research. Protein-coated nanoparticles and virus-like particles (VLP) are among them. Due to their properties, they have potential applications in drug delivery systems and vaccine development. The study focused on modifying the computationally engineered proteins AP01 and TB08 using computationally designed nanoparticles, I3-71 and Cp149. These modified proteins have potential applications in drug delivery systems and other biomedical fields.

Nanoparticles I3-71 and Cp149(Cys150) were modified with TB08 and AP01 forming protein complexes, TB08-S3.1-I3-71 and AP01-S2.3-Cp149-(3 mutations)-Construct C. The modified proteins were expressed individually, in parallel, the modified proteins were co-expressed by being paired with their respective native scaffolds, I3-71 and Cp149(Cys150). The native scaffolds, I3-71 and Cp149(Cys150) were used as positive controls. The modified proteins were purified using immobilized metal affinity chromatography (IMAC), ammonium sulfate precipitation and size exclusion chromatography (SEC). Modified proteins were characterized using SDS-PAGE, SEC and dynamic light scattering (DLS) determining molecular weight and size. 

The expression of TB08-S3.1-I3-01 was not successful as it was not detected by SDS-PAGE or SEC, expression and purification of TB08-S3.1-I3-71 and AP01-S2.3-Cp149(3 mutations)-Construct C was only successful under denaturing conditions. The coexpression of both modified proteins TB08-S3.1-I3-71 with I3-71 and AP01-S2.3-Cp149-(3 mutations)-Construct C with Cp149(Cys150) improved the protein folding as SDS-PAGE and SEC characterized both modified proteins under native conditions. DLS measurement for the coexpressed modified AP01 protein confirmed particle size that resembles the reported theoretical size of Cp149. Unfortunately, DLS could not properly measure coexpressed modified TB08 protein.

This study faced the challenges of expressing, purifying, and characterizing modified AP01 and TB08 proteins. Individually expressed modified proteins endured possible protein misfolding or aggregations, as characterization was impossible. Coexpressed modified proteins implemented the native scaffold, which was used as a positive control, which made characterization possible, most likely due to chaperone-like interactions which assisted with proper protein folding. Implementing methods such as mass spectrometry and cryo-electron microscopy could address characterization issues for future applications.