Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Virulence sensor histidine kinase phoQ
Abbreviated Name	PhoQ-SD
SCOP Family	Unknown
Structure Notes
Organism	Salmonella typhimurium
UniProt Accession	P14147
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Tetramer

Construct
Full Length	n
Domain	146 periplasmic domain
Chimera	n/a
Variants	n/a
Chain Length	156
Molecular Weight	18023.4
Pi	4.77
Molecular Weight	18023.4
Disulphides	Unknown
Full Sequence	VGYSVSFDKTTFRLLRGESNLFYTLAKWENNKISVELPENLDMQSPTMTLIYDETGKLLWTQRNIPWLIKSIQPEWLKTNGFHEIETNVDATSTLLSEDHSAQEKLKEVREDDDDAEMTHSVAVNIYPATARMPQLTIVVVDTIPIELKRSYMVWS
Notes	n/a

Expression
Report	Cho US, Bader MW, Amaya MF, Daley ME, Klevit RE, Miller SI, Xu W. (2006) J Mol Biol, 356, 1193-1206
Project Aim	Undefined
Fusion	N-terminal methionine+C terminal his-tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)
Expression Temp	37.0
Expression Time	5 h
Expression Vector	pETIIa
Expression Protocol	The S. typhimurium PhoQ sensor domain (residues 45–190) with an N-terminal methionine and C-terminal His-tag was cloned into plasmid pET11a and expressed in Escherichia coli BL21(DE3). Expression was induced by the addition of 0.5 mM IPTG at A600=0.6, and cells were grown for an additional 5 h after induction at 37 °C. Cells were then collected by centrifugation, lysed in a French pressure cell, and inclusion bodies were collected by a 15 min spin at 4000g. We chose to purify the PhoQ sensor domain from inclusion bodies for a better protein yield.
Method of Induction	IPTG
Cell Density at Induction	OD 0.6 = 600
Cell Disruption Method	French Press
Lytic Agent	None
Pre-Refolding Purification	not specified
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	n/a
Solubilization Buffer	20 mM sodium phosphate (pH 8.0), 200 mM NaCl, 6 M urea
Refolding Buffer	ice-cold 20 mM sodium phosphate (pH 8.0)
Pre-Refolding Purification	not specified
Tag Cleaved	no
Refolding pH	8.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	The pellet was resuspended in 20 mM sodium phosphate (pH 8.0), 200 mM NaCl, 6 M urea, and incubated for 1 h on ice. Insoluble material was removed by ultracentrifugation at >100,000g. For refolding, the sample was filtered and added to ice-cold 20 mM sodium phosphate (pH 8.0) while stirring the solution. The solution was stirred for another 10 min and then loaded onto a HiTrap column (Amersham Pharmacia) charged with nickel sulfate. The column was extensively washed with 20 mM sodium phosphate (pH 8.0) and with the same buffer containing 40 mM imidazole. Protein was eluted with the same buffer containing 200 mM imidazole. The solublized PhoQ sensor domain after refolding was dialyzed against 50mM Tris–HCl (pH 8.0), 100 mM NaCl, 0.2 mM β-merceptoethanol overnight at 4 °C. The fractions with PhoQ sensor domain were concentrated to 10 mg/ml and further purified with a Superdex-75 size exclusion column pre-equilibrated with 25 mM Tris (pH 8.0), 50 mM NaCl, 1 mM DTT. After the size exclusion column, the protein appears as a single band on SDS-PAGE gel.
Refolding Assay	NMR analysis
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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