Azuma Yusuke, Hilvert Donald
Laboratory of Organic Chemistry, ETH Zurich, Zurich, Switzerland.
Methods Mol Biol. 2018;1798:39-55. doi: 10.1007/978-1-4939-7893-9_4.
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.
将活性酶包裹在蛋白质笼中是一种控制催化活性的有效策略。利用绿色荧光蛋白（GFP）的带正电超荷变体GFP(+36)作为基因可编程标签，酶可以快速、定量地装入嗜热栖热菌笼形蛋白鲁美嗪合酶（AaLS-13）的工程变体中，该变体具有带负电的内腔。只需将各组分在水溶液中混合，货物就会自发地定位在AaLS-13笼内。本章描述了制备AaLS-13笼和GFP(+36)-酶融合体的详细方案以及包合物的表征方法。还讨论了合适的包封条件和酶动力学测定方法。
