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Characterization of the sensor domain of QseE histidine kinase from Escherichia coli.
- Source :
-
Protein expression and purification [Protein Expr Purif] 2016 Oct; Vol. 126, pp. 122-126. Date of Electronic Publication: 2016 Jun 29. - Publication Year :
- 2016
-
Abstract
- In enterohemorrhagic Escherichia coli (EHEC), the QseEF two-component system causes attaching and effacing (AE) lesion on epithelial cells. QseE histidine kinase senses the host hormone epinephrine, sulfate, and phosphate; it also regulates QseF response regulator, which activates LEE gene that encodes AE lesion. In order to understand the recognition of ligand molecules and signal transfer mechanism in pathogenic bacteria, structural studies of the sensor domain of QseE of Escherichia coli should be conducted. In this study, we describe the overexpression, purification, and structural and biophysical properties of the sensor domain of QseE. The fusion protein had a 6×His tag at its N-terminus; this protein was overexpressed as inclusion bodies in E. coli BL21 (DE3). The protein was denatured in 7M guanidine hydrochloride and refolded by dialysis. The purification of the refolded protein was carried out using Ni-NTA affinity column and size-exclusion chromatography. Thereafter, the characteristics of the refolded protein were determined from NMR, CD, and MALS spectroscopies. In a pH range of 7.4-5.0, the folded protein existed in a monomeric form with a predominantly helical structure. (1)H-(15)N HSQC NMR spectra shows that approximately 93% backbone amide peaks are detected at pH 5.0, suggesting that the number of backbone signals is sufficient for NMR studies. These data might provide an opportunity for structural and functional studies of the sensor domain of QseE.<br /> (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Subjects :
- Enterohemorrhagic Escherichia coli genetics
Hydrogen-Ion Concentration
Nuclear Magnetic Resonance, Biomolecular
Protein Domains
Protein Structure, Secondary
Recombinant Fusion Proteins biosynthesis
Recombinant Fusion Proteins chemistry
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins isolation & purification
Enterohemorrhagic Escherichia coli enzymology
Escherichia coli Proteins biosynthesis
Escherichia coli Proteins genetics
Escherichia coli Proteins isolation & purification
Gene Expression
Protein Refolding
Receptors, Adrenergic biosynthesis
Receptors, Adrenergic genetics
Receptors, Adrenergic isolation & purification
Subjects
Details
- Language :
- English
- ISSN :
- 1096-0279
- Volume :
- 126
- Database :
- MEDLINE
- Journal :
- Protein expression and purification
- Publication Type :
- Academic Journal
- Accession number :
- 27371359
- Full Text :
- https://doi.org/10.1016/j.pep.2016.06.012