Refolding Record:

Protein See all refolding records for this protein...
Protein Name	CX3C chemokine fractalkine
Abbreviated Name	CX3CL1
SCOP Family	interleukin 8-like chemokines
Structure Notes
Organism	Human
UniProt Accession	P78423
SCOP Unique ID	n/a
Structure Solved
Class	Alpha+Beta
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	76
Molecular Weight	8639.0
Pi	9.55
Molecular Weight	8639.0
Disulphides	2
Full Sequence	QHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNG
Notes	n/a

Expression
Report	Mizoue LS, Bazan JF, Johnson EC, Handel TM. (1999) Biochemistry, 38, 1402-1414
Project Aim	Structural Studies
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21 pLysS
Expression Temp	37.0
Expression Time	n/a
Expression Vector	pAED4
Expression Protocol	Expression and Purification of FRCD. DNA encoding residues 1-76 of fractalkine was subcloned into the T7-driven expression vector pAED4 (17) and transformed into the Escherichia coli strain BL21/pLysS. Cells were grown at 37 C in MOPS minimal media (18) containing 13C-glucose (2 g/L) and/or 15N-ammonium sulfate (1 g/L), 100 g/mL of ampicillin, and 25 g/mL chloramphenicol. When the cell density reached 0.7 OD600, protein expression was induced by adding isopropyl -D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. The protein was isolated from inclusion bodies as follows: E. coli cell paste from a 1.5 L growth was sonicated in 150 mL of buffer (10 mM potassium phosphate, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, pH 7.0) and centrifuged at 10000 rpm for 20 min.
Method of Induction	IPTG
Cell Density at Induction	OD 0.7 = 600
Cell Disruption Method	Sonication
Lytic Agent	Detergents
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	25 mM potassium phosphate, 0.25% deoxycholate, pH 7.5
Solubilization Buffer	6 M guanidinium chloride
Refolding Buffer	100 mM Tris, 5 mM EDTA, and 0.2 mM oxidized glutathione, 1 mM reduced glutathione, pH 8.0
Pre-Refolding Purification	None
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	overnight
Redox Agent	GSH
Redox Agent Concentration	1 mM,1 mM,1 mM
Refolding Protocol	E. coli cell paste from a 1.5 L growth was sonicated in 150 mL of buffer (10 mM potassium phosphate, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, pH 7.0) and centrifuged at 10000 rpm for 20 min. The cell pellet was washed twice with 50 mL of buffer containing detergent (25 mM potassium phosphate, 0.25% deoxycholate, pH 7.5) followed by two 50 mL washes in the same buffer without detergent. Refolding and oxidation of the two disulfides were achieved by solubilizing the pellet in a minimal amount of 6 M guanidinium chloride and diluting it 100-fold into a redox buffer containing 100 mM Tris, 5 mM EDTA, and 0.2 mM oxidized glutathione, 1 mM reduced glutathione, pH 8.0. After overnight stirring at 4 C, the solution was adjusted to pH 7.0, centrifuged, filtered, and loaded onto a 30 mL Sepharose SP fast flow column. The column was washed with 2-3 bed volumes of 10 mM potassium phosphate, 5 mM EDTA, pH 7.0, and the protein was eluted with a salt gradient of 0.25-0.65 M in the same buffer. Final purification was achieved by reversed-phase HPLC on a Vydac C4 semi-prep column with a gradient of 26.0:73.9:0.1 to 38:61.9:0.1 acetonitrile/H2O/trifluoroacetic acid, followed by lyophilization. Mass spectral analysis indicated that the protein was oxidized and the initiating methionine was retained.
Refolding Assay	Unspecified
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	7 mg/L.
Purity	n/a
Notes	n/a

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