A Protein‐Based Encapsulation System with Calcium‐Controlled Cargo Loading and Detachment

Abstract

Protein-based encapsulation systems have a wide spectrum of applications in targeted delivery of cargo molecules and for chemical transformations in confined spaces. By engineering affinity between cargo and container proteins it has been possible to enable the efficient and specific encapsulation of target molecules. Missing in current approaches is the ability to turn off the interaction after encapsulation to enable the cargo to freely diffuse in the lumen of the container. Separation between cargo and container is desirable in drug delivery applications and in the use of capsids as catalytic nanoparticles. We describe an encapsulation system based on the hepatitis B virus capsid in which an engineered high-affinity interaction between cargo and capsid proteins can be modulated by Ca . Cargo proteins are loaded into capsids in the presence of Ca , while ligand removal triggers unbinding inside the container. We observe that confinement leads to hindered rotation of cargo inside the capsid. Application of the designed container for catalysis was also demonstrated by encapsulation of an enzyme with β-glucosidase activity.

Shining Light on Polymeric Drug Nanocarriers with Fluorescence Correlation Spectroscopy

“…Such studies are not limited to polymeric NCs, but can also be applied to other nanocarrier system. In particular, nanocarriers based on lipids [40,68,81,95,96,[152][153][154][155][156] and proteins [157][158][159][160] as well as on metal-organic framework, [86,161,162] calcium phosphate, [163,164] silica [165][166][167] or gold [168,169] nanoparticles, were often characterized with FCS. Furthermore, inherently fluorescent nanoparticles such as quantum dots, [101,104,109,[170][171][172] carbon dots [173][174][175] or nanodiamonds [176][177][178][179] have been extensively studied with respect to their interactions with plasma proteins and use for transfection or bioimaging.…”

Protein Cages: From Fundamentals to Advanced Applications

“…176 Another engineered protein−protein interaction based on calmodulin has been employed in HBV capsids. 875 Genetic fusion of the C-terminal domain of calmodulin to cargo proteins and a fragment of the plasma membrane calcium pump to the capsid protein afforded a highaffinity driving force for encapsulation. Interestingly, in this case, the interaction can be disrupted by the addition of Ca 2+ , providing a means to release the encapsulated enzymes on demand.…”

“…For example, α-helical peptides that dimerize with a partner by the formation of a coiled-coil can be appended to internally exposed C- or N-termini. , Fusion of the partner peptide to the desired cargo protein provides a simple means to direct encapsulation of the guest (Figure d). ,,,, An advantage of this in vitro assembly process is that the stoichiometry of the capsid–enzyme complex can be controlled by the input ratio of the components, enabling studies of the effect of loading on catalyst performance . Another engineered protein–protein interaction based on calmodulin has been employed in HBV capsids . Genetic fusion of the C-terminal domain of calmodulin to cargo proteins and a fragment of the plasma membrane calcium pump to the capsid protein afforded a high-affinity driving force for encapsulation.…”

Organizing Multi-Enzyme Systems into Programmable Materials for Biocatalysis

“…One issue of VLPs and other compartmentalized systems is that the cargo proteins often remain attached to the shell interiors due to the encapsulation mechanisms used, limiting dense cargo packaging. To alleviate this issue, a calcium-dependent cargo release system was recently engineered for HBV VLPs [173]. Other controllable protein affinity interactions, e.g., the above mentioned SpyTag/SpyDock system, could be similarly adopted for this purpose.…”