Refolding Record:

Protein See all refolding records for this protein...
Protein Name	C-C motif chemokine 4 Also known as MIP-1-beta
Abbreviated Name	MIP-1beta
SCOP Family	interleukin 8-like chemokines
Structure Notes
Organism	Human
UniProt Accession	P13236
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	67
Molecular Weight	7650.6
Pi	4.76
Molecular Weight	7650.6
Disulphides	Unknown
Full Sequence	MGSDPPTACCFSYTARKLPRNFVVDYYETSSLCSQPAVVFQTKRSKQVCADPSESWVQEYVYDLELN
Notes	n/a

Expression
Report	Jin H, Hayes GL, Darbha NS, Meyer E, LiWang PJ. (2005) Biochemical and Biophysical Research Com, 338, 987-999
Project Aim	Undefined
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	None
Expression Strain	None
Expression Temp	0.0
Expression Time	00
Expression Vector	n/a
Expression Protocol	Protein expression and purification. For wild type proteins and MIP-1β variants and IL-8 variants from the mutagenesis, protein expression and purification followed a standard chemokine refolding and purification method published previously [23]
Method of Induction	Not Stated
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Not stated
Lytic Agent	None
Pre-Refolding Purification	Metal affinity chromatography
Solubility	insoluble

Refolding
Refolding Method	Dilution/Column Refolding Combination
Wash Buffer	n/a
Solubilization Buffer	6 M guanidine chloride, 20 mM sodium phosphate buffer, pH 7.2, containing 250 mM NaCl and 10 mM 2-mercaptoethanol,
Refolding Buffer	decrease the amount of guanidine chloride in 20 mM sodium phosphate buffer, pH 7.5, containing 250 mM NaCl and then eluted with imidazole
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	no tag
Refolding pH	7.5
Refolding Temperature	0.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	For the cI fusion mutants, a slight variation in the procedure was used, in that the proteins were expressed in rich medium, and the protein inclusion body was taken up in 50 ml of 6 M guanidine chloride, 20 mM sodium phosphate buffer, pH 7.2, containing 250 mM NaCl and 10 mM 2-mercaptoethanol, and loaded onto a 5 ml chelating column (Amersham–Pharmacia Biotech) equilibrated with nickel sulfate and the guanidinium buffer. The protein was refolded on the column using a gradient to slowly decrease the amount of guanidine chloride in 20 mM sodium phosphate buffer, pH 7.5, containing 250 mM NaCl and then eluted with imidazole. The fractions containing refolded protein were pooled together and directly diluted 10-fold into 10% acetonitrile and 0.1% trifluoroacetic acid solution, and loaded onto an equilibrated C4 reversed phase column (Vydac, Hesperia, CA). The protein was eluted using an acetonitrile/0.1% TFA gradient. Fractions containing the proteins were quickly frozen in liquid nitrogen and lyophilized by the Labconco freeze dry system (Labconco) into dried powder. A small amount of the fractions of each run was collected and run on 12% SDS–PAGE to confirm the protein purity.
Refolding Assay	Unspecified
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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