Purification and Characterization of Glutathione Binding Protein GsiB from Escherichia coli.

Zhongshan Wang,Yanqiang Li,Meixian Zhang,Jiawei Fang,Xiaokun Xia,Meng Zhang

doi:10.1155/2018/3429569

Abstract

Objectives To purify and characterize the glutathione binding protein GsiB of glutathione importer (GSI) in Escherichia coli (E. coli). Results The coding sequence of GsiB was cloned from E. coli MG1655 and expressed in BL21(DE3). GsiB protein was expressed and purified to homogeneity using Ni-affinity and gel filtration chromatography. SDS-PAGE of purified GsiB showed a single protein band of molecular mass 56 kDa, while native gel showed two bands around 56 kDa and 110 kDa. Gene knockout showed that GsiB was essential for GSI mediated glutathione import. Interactions of GsiA, B, C, and D were determined using bacterial two-hybrid method. Without glutathione, GsiB showed no direct interaction with the other three proteins. However, GsiB could interact with GsiC and GsiD when using glutathione as sole sulfur source. Conclusions GsiB functions in E. coli was characterized which could help elucidate the glutathione import mechanism in gram-negative bacteria.

Highlights

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine; glutathione manifests predominantly in thiol-reduced form (GSH)) is the most important endogenous antioxidant across all kingdoms of life
The intracellular GSH homeostasis can be maintained by different pathways, including de-novo synthesis, GSH redox cycling, and direct uptake [9]
GSH import by bacteria may serve as organic sulfur resource [10,11,12]

Summary

Introduction

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine; GSH) is the most important endogenous antioxidant across all kingdoms of life. GSH is a tripeptide consisting of glutamate, cysteine, and glycine amino acids. GSH is distributed ubiquitously and usually attains mM concentrations in human body. It plays an important role in maintaining the intracellular redox homeostasis [1], as well as in the detoxification of xenobiotics and their metabolites [2, 3]. GSH functions in salvage of cysteine [4] and cell signaling [5, 6]. Glutathione manifests predominantly in thiol-reduced form (GSH) [7]. The intracellular GSH homeostasis can be maintained by different pathways, including de-novo synthesis, GSH redox cycling, and direct uptake [9]. The mechanisms that underpin glutathione uptake still need further investigation

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Methods

Conclusion