Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Multiple antibiotic resistance protein marA
Abbreviated Name	MarA
SCOP Family	AraC type transcriptional activator [46759]
Structure Notes
Organism	Escherichia coli
UniProt Accession	P0ACH5
SCOP Unique ID	n/a
Structure Solved
Class	Alpha
Molecularity	Monomer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	127
Molecular Weight	15184.2
Pi	9.36
Molecular Weight	15184.2
Disulphides	Unknown
Full Sequence	MSRRNTDAITIHSILDWIEDNLESPLSLEKVSERSGYSKWHLQRMFKKETGHSLGQYIRSRKMTEIAQKLKESNEPILYLAERYGFESQQTLTRTFKNYFDVPPHKYRMTNMQGESRFLHPLNHYNS
Notes	n/a

Expression
Report	Jair KW, Martin RG, Rosner JL, Fujita N, Ishihama A, Wolf RE Jr. (1995) J Bacteriol., 177, 7100-7104
Project Aim	Purification & characterization
Fusion	N-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)
Expression Temp	34.0
Expression Time	3 h
Expression Vector	pET15b
Expression Protocol	Purification of MarA. Strain N8224 was grown in 2 liters of superbroth at 37􏰝C to an A600 of 0.8. IPTG (isopropyl-􏱇-D-thiogalactopyranoside) was then added to 0.4 mM, and vigorous aeration was continued for 3 h. The remaining steps were carried out in the cold. The cells were harvested by centrifugation (5 g [wet weight]), washed with 25 ml of 50 mM Tris-HCl (pH 7.5)–1 mM EDTA–1 M NaCl, frozen overnight, and resuspended in 25 ml of the bufferVOL.
Method of Induction	IPTG
Cell Density at Induction	OD 0.8 = 600
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	not specified
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	25 ml of 50 mM Tris-HCl (pH 7.5)–1 mM EDTA–1 M NaCl
Solubilization Buffer	25 ml of 50 mM Tris-HCl (pH 8.5)–6 M guanidinium-HCl
Refolding Buffer	50 mM Tris-HCl (pH 8.5)
Pre-Refolding Purification	not specified
Tag Cleaved	no
Refolding pH	8.5
Refolding Temperature	0.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	The cells were sonically disrupted with eight 15-s bursts and centrifuged at 120,000 g for 30 min. The supernatant fluid was discarded, the pellet was rinsed with 30 ml of 4 M urea–50 mM Tris-HCl (pH 8.5), and centrifuged again at 120,000 g for 30 min. Finally, the pellet was resuspended in 25 ml of 50 mM Tris-HCl (pH 8.5)–6 M guanidinium-HCl and centrifuged for 30 min at 120,000 g. The resulting solution was diluted to 75 ml with 50 mM Tris-HCl (pH 8.5) and passed through a 100-ml bed volume of chelating Sepharose equilibrated with 0.1 M NiCl2 that had been washed three times with water. The column was eluted with a linear gradient of 0 to 1.0 M imidazole in 1 M NaCl–50 mM Tris-HCl (pH 8.5). A sharp peak of MarA protein eluting at 0.2 M imidazole was collected and extensively dialyzed against buffer A (1 M NaCl–50 mM HEPES [N-2-hydroxyethylpipera- zine-N-2-ethanesulfonic acid; pH 8.0]–1 mM dithiothreitol–5 mM EDTA–0.1 mM Triton X-100). The protein was concentrated with Amicon membranes to 45 ml (0.6 mg/ml) and digested with 500 U of thrombin for 1 h at 0C. The thrombin was removed by benzamidine Sepharose chromatography, and the histidine tag was removed by Sephadex gel filtration, leaving the MarA protein as wild type except for one N-terminal histidine residue from the expression vector. After extensive dialysis against buffer A in dialysis tubing with a molecular weight exclusion of 8,000, the protein was again concentrated with Amicon membranes and finally was dialyzed against buffer A or buffer A supplemented with 20% glycerol.
Refolding Assay	SDS-PAGE
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

Back to refolding records...