Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY, 40536, USA; West China School of Pharmacy, Sichuan University, No. 17 People's South Road, Chengdu, 610041, PR China.
Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY, 40536, USA.
Chem Biol Interact. 2019 Sep 1;310:108756. doi: 10.1016/j.cbi.2019.108756. Epub 2019 Jul 17.
Human butyrylcholinesterase (BChE) is a widely distributed plasma enzyme. For decades, numerous research efforts have been directed at engineering BChE as a bioscavenger of organophosphorus insecticides and chemical warfare nerve agents. However, it has been a grand challenge to cost-efficiently produce BChE in large-scale. Recently reported studies have successfully designed a truncated BChE mutant (with amino-acid substitutions on 47 residues that are far away from the catalytic site), denoted as BChE-M47 for convenience, which can be expressed in E. coli without loss of its catalytic activity. In this study, we aimed to dimerize the truncated BChE mutant protein expressed in a prokaryotic system (E. coli) in order to further improve its thermal stability by introducing a pair of cross-subunit disulfide bonds to the BChE-M47 structure. Specifically, the E377C/A516C mutations were designed and introduced to BChE-M47, and the obtained new protein entity, denoted as BChE-M48, with a pair of cross-subunit disulfide bonds indeed exists as a dimer with significantly improved thermostability and unaltered catalytic activity and reactivity compared to BChE-M47. These results provide a new strategy for optimizing protein stability for production in a cost-efficient prokaryotic system. Our enzyme, BChE-M48, has a half-life of almost one week at a 37°C, suggesting that it could be utilized as a highly stable bioscavenger of OP insecticides and chemical warfare nerve agents.
人丁酰胆碱酯酶（BChE）是一种广泛分布的血浆酶。几十年来，人们一直致力于将 BChE 工程化为有机磷杀虫剂和化学战剂的生物清除剂。然而，高效地大规模生产 BChE 一直是一个巨大的挑战。最近的研究报告成功设计了一种截断的 BChE 突变体（在远离催化位点的 47 个氨基酸上进行了氨基酸取代），为方便起见，将其表示为 BChE-M47，它可以在不丧失其催化活性的情况下在大肠杆菌中表达。在这项研究中，我们旨在将在原核系统（大肠杆菌）中表达的截断的 BChE 突变体蛋白二聚化，通过在 BChE-M47 结构中引入一对交叉亚基二硫键来进一步提高其热稳定性。具体来说，设计并引入了 E377C/A516C 突变，得到的新蛋白实体，记为 BChE-M48，确实存在一对交叉亚基二硫键，作为二聚体存在，与 BChE-M47 相比，具有显著提高的热稳定性、不变的催化活性和反应性。这些结果为在高效的原核系统中生产优化蛋白质稳定性提供了一种新策略。我们的酶 BChE-M48 在 37°C 下的半衰期几乎为一周，表明它可以用作 OP 杀虫剂和化学战剂的高度稳定的生物清除剂。
