Abstract
The packaging of active enzymes in protein cages is a powerful strategy to control catalytic activity. Using a positively supercharged variant of green fluorescent protein, GFP(+36), as a genetically programmable tag, enzymes can be rapidly and quantitatively loaded into an engineered variant of the Aquifex aeolicus cage-forming protein lumazine synthase (AaLS-13) that possesses a negatively charged lumen. The cargo is spontaneously localized within AaLS-13 cages by simply mixing the components in aqueous solution. This chapter describes a detailed protocol for the preparation of AaLS-13 cages and GFP(+36)-enzyme fusions, as well as characterization of the inclusion complexes. Suitable conditions for encapsulation and enzyme kinetic assays are also discussed.
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