Thermostability and molecular encapsulation within an engineered caged protein scaffold

Abstract

Self-assembling biological complexes such as viral capsids have been manipulated to function in innovative nanotechnology applications. The E2 component of pyruvate dehydrogenase from Bacillus stearothermophilus forms a dodecahedral complex and potentially provides another platform for these purposes. In this investigation, we show that this protein assembly exhibits unusual stability and can be modified to encapsulate model drug molecules. To distill the E2 protein down to its structural scaffold core, we synthesized a truncated gene optimized for expression in Escherichia coli. The correct assembly and dodecahedral structure of the resulting scaffold was confirmed with dynamic light scattering and transmission electron microscopy. Using circular dichroism and differential scanning calorimetry, we found the thermostability of the complex to be unusually high, with an onset temperature of unfolding at 81.1 +/- 0.9 degrees C and an apparent midpoint unfolding temperature of 91.4 +/- 1.4 degrees C. To evaluate the potential of this scaffold for encapsulation of guest molecules, we made variants at residues 381 and 239 which altered the physicochemical properties of the hollow internal cavity. These mutants, yielding 60 and 120 mutations within this cavity, assembled into the correct architecture and exhibited high thermostability that was comparable to the wild-type scaffold. To show the applicability of this scaffold, two different fluorescent dye molecules were covalently coupled to the cysteine mutant at site 381. We demonstrate that these mutations can introduce non-native functionality and enable molecular encapsulation within the cavity while still retaining the dodecahedral structure. The unusually robust nature of this scaffold and its amenability to internal changes reveal its potential for nanoscale applications.

Viral-mimicking protein nanoparticle vaccine for eliciting anti-tumor responses

“…1C) that are within the reported optimal range for viral-based vaccines [17]. These nanoparticle diameters are also consistent with sizes observed for our conjugation with other peptides and guest molecules [26,29,30,34], further demonstrating the versatility of the E2 platform for attachment of various molecules ( e.g., epitopes).…”

“…The D381C E2 protein nanoparticle (E2) was prepared and characterized as previously described [26,30]. D381C is an E2 mutant with a non-native cysteine introduced to the internal cavity of the nanoparticle at amino acid location 381 for site-specific conjugation.…”

“…DLS was used to measure hydrodynamic diameters, and transmission electron micrographs of 2% uranyl acetate-stained nanoparticle on Cu 150 mesh Formvar-carbon coated grids were obtained on a JEM1200EX (JEOL) with a Bioscan600W digital camera (Gatan) [26,30]. …”

“…The E2 nanoparticle is a self-assembling hollow protein cage with an approximately 30-nm diameter and high physical stability [26]. It is also of non-viral origin and has been shown to be amenable to functionalization in biomedical applications [26–29].…”

“…The E2 nanoparticle is a self-assembling hollow protein cage with an approximately 30-nm diameter and high physical stability [26]. It is also of non-viral origin and has been shown to be amenable to functionalization in biomedical applications [26–29]. Our research group has previously demonstrated significantly enhanced activation and cross-presentation of a model antigen using the E2 nanoparticle for delivery to and activation of DCs [30].…”