Enzyme Encapsulation in an Engineered Lumazine Synthase Protein Cage
Enzyme Encapsulation in an Engineered Lumazine Synthase Protein Cage
复制标题
DOI：
10.1007/978-1-4939-7893-9_4
复制
发表时间：
2018-01-01
期刊：
影响因子：
--
通讯作者：
Hilvert, Donald
中科院分区：
文献类型：
--
作者：
Azuma, Yusuke;Hilvert, Donald
将活性酶包装在蛋白笼中是控制催化活性的有力策略。使用带正电的绿色荧光蛋白 GFP(+36) 变体作为基因可编程标签，可以将酶快速定量地加载到具有带负电腔的 Aquifex aeolicus 笼形成蛋白二氧四氢呋喃合酶 (AaLS-13) 的工程变体中。通过简单地将各成分混合在水溶液中，货物就会自发地定位在 AaLS-13 笼内。本章描述了 AaLS-13 笼和 GFP(+36)-酶融合物的制备的详细方案，以及包合复合物的表征。还讨论了适合封装和酶动力学测定的条件。
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.