Markova Svetlana V, Larionova Marina D, Vysotski Eugene S
Photobiology Laboratory, Institute of Biophysics SB RAS, Federal Research Center "Krasnoyarsk Science Center SB RAS", Krasnoyarsk, Russia.
School of Fundamental Biology and Biotechnology, Siberian Federal University, Krasnoyarsk, Russia.
Methods Mol Biol. 2022;2524:59-73. doi: 10.1007/978-1-0716-2453-1_5.
The small coelenterazine-dependent luciferase from Metridia longa (MLuc), in view of its high activity, simplicity of bioluminescent (BL) reaction, and stability, has found successful analytical applications as a genetically encoded reporter for in vivo assessment of cellular processes. However, the study on the biochemical and BL properties and the development of in vitro analytical applications of MLuc are hampered by the difficulties of obtaining a sufficient amount of the highly active recombinant protein due to the presence of multiple (up to five) disulfide bonds per molecule. Here, we present a protocol to obtain the recombinant disulfide-rich MLuc using a cheap and simple Escherichia coli expression system without any affinity tags in its native form by refolding from inclusion bodies. The method includes (i) purification of MLuc inclusion bodies, solubilization of the aggregated form with full reduction of disulfide bonds, and refolding to the native state using a glutathione redox system in the presence of arginine and Cu ions and (ii) chromatographic purification of MLuc and its functional assessment in terms of activity. We introduce the empirical, optimal conditions for oxidative refolding and subsequent purification of MLuc, with its basic properties taken into account. We believe that this protocol is adaptable for a large-scale harvest of other natively folded copepod luciferases as well as other disulfide-rich recombinant proteins from E. coli inclusion bodies.
来自长腹水蚤（MLuc）的小型腔肠素依赖性荧光素酶，鉴于其高活性、生物发光（BL）反应的简单性和稳定性，已成功用作细胞过程体内评估的基因编码报告分子，在分析应用中取得了成功。然而，由于每个分子存在多个（多达五个）二硫键，难以获得足够量的高活性重组蛋白，这阻碍了对MLuc的生化和BL特性的研究以及其体外分析应用的开发。在此，我们提出了一种方案，使用廉价且简单的大肠杆菌表达系统，通过从包涵体中重折叠，以天然形式获得无任何亲和标签的富含二硫键的重组MLuc。该方法包括：（i）纯化MLuc包涵体，用完全还原二硫键的方法溶解聚集形式，并在精氨酸和铜离子存在下使用谷胱甘肽氧化还原系统将其重折叠至天然状态；（ii）对MLuc进行色谱纯化并根据活性对其进行功能评估。我们介绍了氧化重折叠和随后纯化MLuc的经验性最佳条件，同时考虑了其基本特性。我们相信，该方案适用于大规模收获其他天然折叠的桡足类荧光素酶以及来自大肠杆菌包涵体的其他富含二硫键的重组蛋白。
