Cloning, overexpression, and purification of glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa
Cloning, overexpression, and purification of glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa
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DOI：
10.1016/j.pep.2017.10.004
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发表时间：
2018-02-01
影响因子：
1.6
通讯作者：
Velasco-Garcia, Roberto
中科院分区：
文献类型：
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作者：
Acero-Navarro, Kevin E.;Jimenez-Ramirez, Mariella;Velasco-Garcia, Roberto
葡萄糖-6-磷酸脱氢酶（G6PDH） （EC 1.1.1.363）在人类病原体铜绿假单胞菌中发挥重要作用，因为它产生NADPH，这是几种生物合成途径和抗氧化酶的必需辅助因子。P. aeruginosa G6PDH也是多种碳源代谢的关键酶，如葡萄糖、甘油、果糖和甘露醇。了解控制该酶活性的动力学特征和机制对今后的研究至关重要。然而，实现这一目标的障碍之一是当前纯化方案实施时获得的蛋白质数量有限，这是限制其生化表征的一个因素。在这项研究中，我们报告了一种快速、高效、可重复的纯化铜绿假单胞菌G6PDH的方法，该方法可以在短时间内（2天）实现。为了建立该方案，克隆了编码该酶的zwf基因，并在大肠杆菌细胞中过表达。与其他方法相比，我们的方法是基于CaCl2的蛋白质沉淀和离子交换色谱的进一步纯化。使用该方案，我们能够获得31 mg/L的纯蛋白，其比活性为145.7 U/mg。本研究获得的重组酶与前人报道的该分子具有相似的物理化学和动力学性质。利用该方法获得的大量活性酶将有助于其结构表征，并识别铜绿假单胞菌和人类G6PDH之间的差异，从而有助于寻找针对细菌酶的选择性抑制剂。(C) 2017爱思唯尔公司版权所有。
Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.363) plays an important role in the human pathogen Pseudomonas aeruginosa because it generates NADPH, an essential cofactor for several biosynthetic pathways and antioxidant enzymes. P. aeruginosa G6PDH is also a key enzyme in the metabolism of various carbon sources, such as glucose, glycerol, fructose, and mannitol. Understanding the kinetic characteristics and mechanisms that control the activity of this enzyme is crucial for future studies in this context. However, one of the impediments to achieving this goal is the limited amount of protein obtained when current purification protocols are implemented, a factor curtailing its biochemical characterization. In this study, we report a fast, efficient and reproducible procedure for the purification of P. aeruginosa G6PDH that can be implemented in a short period (2 days). In order to establish this protocol, the zwf gene, which encodes for this enzyme, was cloned and overexpressed in Escherichia coli cells. In contrast to other procedures, our method is based on protein precipitation with CaCl2 and further purification by ion exchange chromatography. Using this protocol, we were able to obtain 31 mg/L of pure protein that manifested specific activity of 145.7 U/mg. The recombinant enzyme obtained in this study manifested similar physicochemical and kinetic properties to those reported in previous works for this molecule. The large quantities of active enzyme obtained using this procedure will facilitate its structural characterization and identify differences between P. aeruginosa- and human G6PDH, thus contributing to the search for selective inhibitors against the bacterial enzyme. (C) 2017 Elsevier Inc. All rights reserved.