Refolding Record:

Protein See all refolding records for this protein...
Protein Name	CC Chemokine RANTES
Abbreviated Name	CCL5
SCOP Family	interleukin 8-like chemokines
Structure Notes
Organism	Human
UniProt Accession	P13501
SCOP Unique ID	n/a
Structure Solved
Class	Alpha+Beta
Molecularity	Dimer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	92
Molecular Weight	9989.7
Pi	9.23
Molecular Weight	9989.7
Disulphides	2
Full Sequence	MKVSAAALAVILIATALCAPASASPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMS
Notes	n/a

Expression
Report	Veldkamp CT, Peterson FC, Hayes PL, Mattmiller JE, Haugner JC 3rd, de la Cruz N, Volkman BF. (2007) Protein Expression and Purification, 52, 202-209
Project Aim	NMR
Fusion	N-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	SG13009 [pREP4]
Expression Temp	37.0
Expression Time	6 h
Expression Vector	pQE30
Expression Protocol	Escherichia coli strain SG13009[pREP4] containing the SDF-1α expression plasmid was grown at 37 °C in either 1 L of Luria-Bertani or M9 minimal media containing 15NH4Cl as the sole nitrogen source. Once an OD600 of 0.7 was reached expression was induced through the addition of 1 mM IPTG. After 6 h cells were harvested by centrifugation at 5000g and stored at −80 °C.
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.7
Cell Disruption Method	French Press
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Column refolding: Nickel-chelating chromatography
Wash Buffer	50 mM sodium phosphate pH 7.4, 300 mM NaCl, 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride, and 0.1% (v/v) 2-mercaptoethanol
Solubilization Buffer	50 mM sodium phosphate pH 7.4, 300 mM NaCl, 10 mM imidazole, and 6 M guanidinium hydrochloride
Refolding Buffer	20 mM Tris pH 8.0, 100 mM NaCl, 5 mM β-cyclodextrin, 1 mM reduced glutathione, 0.5 mM oxidized glutathione
Pre-Refolding Purification	None
Tag Cleaved	no
Refolding pH	8.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	GSH/GSSG
Redox Agent Concentration	n/a,1 / 0/5 mM,1 / 0.5 mM
Refolding Protocol	Protein purification and on-column refolding The procedures for solution and on-column refolding of SDF-1α are the same up to the point of batch loading the Ni sepharose™ 6 column and the 30 min incubation. After incubation, the column was washed with 100 mL of detergent buffer (20 mM Tris pH 8.0, 100 mM NaCl, 1% Triton X-100 (v/v), 10 mM 2-mercaptoethanol) followed by 100 mL of oxidation buffer (20 mM Tris pH 8.0, 100 mM NaCl, 5 mM β-cyclodextrin, 1 mM reduced glutathione, 0.5 mM oxidized glutathione) and 70 mL of wash buffer (20 mM Tris pH 8.0, 500 mM NaCl). The refolded SDF-1α containing correctly oxidized disulfide bonds was eluted with elution buffer (25 mM Hepes pH 7.4, 300 mM NaCl, 1 M imidazole). Fractions containing SDF-1α were pooled and dialyzed against 4 L of 0.3% acetic acid and then lyophilized for CNBr cleavage. MALDI-TOF MS analysis of the SDF-1α was done before and after the CNBr cleavage to check for hexahistadine tag removal. After the CNBr cleavage the refolded, native N-terminal SDF-1α was lyophilized and purified to >98% homogeneity by RP HPLC. On-column refolding and purification of RANTES/CCL5, lymphotactin/XCL1, and fractalkine/CX3CL1 were performed in a similar fashion.
Refolding Assay	Unspecified
Refolding Chaperones	None
Refolding Additives	None,β-cyclodextrin
Additives Concentration	5 mM
Refolding Yield	n/a
Purity	n/a
Notes	Refolding /purification protocol for SDF-1a and CC chemokine rantes is same.

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