Purification and Characterization of Glutathione Binding Protein GsiB from Escherichia coli
Zhongshan Wang1, Xiaokun Xia2, Meixian Zhang3
1Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou, China.
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.
Related Concept Videos
Factors Affecting Protein-Drug Binding: Protein-Related Factors
The physicochemical properties of a drug play a significant role in its ability to bind to proteins. Lipophilic drugs, which dissolve in fats, oils, and lipids, can be...
Detergent Purification of Membrane Proteins
The Equilibrium Binding Constant and Binding Strength
Drug Distribution: Plasma Protein Binding
Protein-Drug Binding: Determination Methods
Indirect methods involve isolating the bound drug from its free form in biological samples such as blood, serum, or plasma. These techniques aim to measure the percentage of drugs bound to proteins. Equilibrium dialysis is a commonly used method where the free drug concentration at equilibrium is measured by separating the bound...
Single-Strand DNA Binding Proteins


