Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Calcium-binding protein C also known as Calcium-binding protein 3
Abbreviated Name	CBP3
SCOP Family	Unknown
Structure Notes
Organism	Dictyostelium discoideum
UniProt Accession	Q54QT7
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	166
Molecular Weight	19085.5
Pi	5.49
Molecular Weight	19085.5
Disulphides	Unknown
Full Sequence	MLTNNEIYQEVQKFAKGYDLNQDGVLTSQEIYYSLLKKMNGNSYEASKATGVLCSTIDINKDGKFSYHEIAKYCADNAKKLIEQNADIAALADVEAFLLRFDKDKDRKLNKTEFVEYFKGGTDTPYSDRDYVLKIIDLDKDGCVSANELQEWFKKKRIDYASRPHC
Notes	n/a

Expression
Report	Lee CH, Jeong SY, Kim BJ, Choi CH, Kim JS, Koo BM, Seok YJ, Yim HS, Kang SO. (2005) Biochim Biophys Acta., 1743, 281-290
Project Aim	Undefined
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)pLysS
Expression Temp	37.0
Expression Time	3 h
Expression Vector	pET-CBP3
Expression Protocol	E. coli BL21 (DE3) pLysS cells harboring pET-CBP3 was grown in an LB medium containing ampicillin. Cells were grown to OD600 of 0.5 at 37 °C and expression was induced by 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). After a 3 h induction, the cells were collected and stored at −20 °C. The frozen cells were thawed and suspended in a binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris–HCl, pH 7.9) and were gently sonicated and centrifuged at 12,000 rpm for 15 min.
Method of Induction	IPTG
Cell Density at Induction	OD 0.5 = 600
Cell Disruption Method	Freeze/Thaw+Sonication
Lytic Agent	None
Pre-Refolding Purification	Metal affinity chromatography
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	n/a
Solubilization Buffer	5 mM imidazole, 0.5 M NaCl, 20 mM Tris–HCl, pH 7.9, 6 M urea
Refolding Buffer	20 mM Tris–HCl, pH 8.5, 30 mM l-arginine, 0.5 mM oxidized glutathione, 150 mM NaCl, 2.5 mM CaCl2
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	no
Refolding pH	8.5
Refolding Temperature	25.0
Protein Concentration	10 uM
Refolding Time	2 h
Redox Agent	GSSG
Redox Agent Concentration	0.5 mM
Refolding Protocol	The pellets were solubilized in a binding buffer, which contained 6 M urea and purified using Ni-affinity column under the denaturation condition (Novagen). The eluents were strongly reduced by adding DTT to a final concentration of 20 mM and refolded rapidly by 100-fold dilution into a refolding buffer (20 mM Tris–HCl, pH 8.5, 30 mM l-arginine, 0.5 mM oxidized glutathione, 150 mM NaCl, 2.5 mM CaCl2). The concentration of the protein during the refolding process was approximately 10 μM. The samples were incubated at room temperature for 2 h and concentrated by ultrafiltration (Amicon). The N-terminal His-tag leader was removed by thrombin protease. The cleavage of the His-tag leader and the purity of the protein were assessed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of the protein was measured using DC protein assay kit (Bio-Rad).
Refolding Assay	SDS-PAGE,Phototaxis assay
Refolding Chaperones	None
Refolding Additives	L-Arginine
Additives Concentration	30 mM
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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