RESUMO
Porous protein crystals provide a template for binding and organizing guest macromolecules. Peroxidase, oxidase, and reductase enzymes immobilized in protein crystals retained activity in single-crystal and bulk assay formats. Several binding strategies, including metal affinity and physical entrapment, were employed to encourage enzyme adsorption into the protein crystals and to retain the enzymes for multiple recycles. Immobilized enzymes had lower activity compared to free enzyme in solution, in part due to diffusion limitations of substrate within the crystal pores. However, the immobilized enzymes were long-term stable and showed increased thermal tolerance. The potential applications of enzyme-laden crystals as sensing devices, delivery capsules, and microreactors motivate future development of this technology.
Assuntos
Proteínas de Bactérias/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Aspergillus niger/enzimologia , Campylobacter jejuni , Difusão , Glucose Oxidase/química , Glucose Oxidase/metabolismo , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , PorosidadeRESUMO
The binding and release of guest fluorescent proteins inside a protein crystal with 13 nm axial pores is controlled. Spatially segregated guest protein loading is achieved via sequential binding and release stages. Additionally, selective stabilization of the crystal exterior results in hollow crystalline shells.
Assuntos
Proteínas de Bactérias/química , Campylobacter jejuni/metabolismo , Cristalização , Corantes Fluorescentes/química , Fatores de TempoRESUMO
DNA assemblies have been used to organize inorganic nanoparticles into 3D arrays, with emergent properties arising as a result of nanoparticle spacing and geometry. We report here the use of engineered protein crystals as an alternative approach to biologically mediated assembly of inorganic nanoparticles. The protein crystal's 13 nm diameter pores result in an 80% solvent content and display hexahistidine sequences on their interior. The hexahistidine sequence captures Au25(glutathione)â¼17 (nitrilotriacetic acid)â¼1 nanoclusters throughout a chemically crosslinked crystal via the coordination of Ni(ii) to both the cluster and the protein. Nanoparticle loading was validated by confocal microscopy and elemental analysis. The nanoparticles may be released from the crystal by exposure to EDTA, which chelates the Ni(ii) and breaks the specific protein/nanoparticle interaction. The integrity of the protein crystals after crosslinking and nanoparticle capture was confirmed by single crystal X-ray crystallography.
