Refolding Record:

Protein See all refolding records for this protein...
Protein Name	human granulocyte-macrophage colony-stimulating factor
Abbreviated Name	hG-CSF
SCOP Family	Long-Chain Cytokines
Structure Notes
Organism	Human
UniProt Accession	P09919
SCOP Unique ID	n/a
Structure Solved
Class	Alpha
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	181
Molecular Weight	19058.1
Pi	5.43
Molecular Weight	19058.1
Disulphides	2
Full Sequence	ATPLGPASSLPQSFLLKCLEQVRKIQGDGAA LQEKLVSECATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQG LLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRR AGGVLVASHLQSFLEVSYRVLRHLAQP
Notes	n/a

Expression
Report	Wang C, Wang L, Geng X (2007) Biomedical chromatography, 1, in press
Project Aim	Protein refolding
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	DH5α
Expression Temp	30.0
Expression Time	4 h
Expression Vector	pBV220
Expression Protocol	Expression of rhG-CSF and solubilization of inclusion bodies. rhG-CSF was expressed in E. coli, cells were disrupted by sonication on an ice bath, then inclusion bodies were collected by centrifugation and solubilized in 8.0 mol/L urea solution containing 0.05 mol/L Tris (pH 8.0), 1 mmol/L EDTA and 0.1 mol/L β-mercaptoethanol following reference (Wang et al., 2006).
Method of Induction	IPTG
Cell Density at Induction	OD 1 = 600
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	0.05 mol/L Tris, pH 8.0, 3.0 mol/L urea, 1.0 mmol/L EDTA, 2.5 mmol/L GSH and 0.8 mmol/L GSSG
Solubilization Buffer	8.0 mol/L urea solution containing 0.05 mol/L Tris (pH 8.0), 1 mmol/L EDTA and 0.1 mol/L β-mercaptoethanol
Refolding Buffer	0.05 mol/L Tris, pH 8.0, 1.0 M NaCl, 3.0 mol/L urea, 1.0 mmol/L EDTA, 2.5 mmol/L GSH and 0.8 mmol/L GSSG
Pre-Refolding Purification	None
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	25.0
Protein Concentration	2.8 mg/ml
Refolding Time	n/a
Redox Agent	GSH/GSSG
Redox Agent Concentration	2.5/0.8 mM,2.5/0.8 mM,2.5/0.8 mM,2.5/0.8 mM,2.5/0.8 mM
Refolding Protocol	refolding of rhG-CSF by dilution. To run a blank gradient elution without injection sample under the same chromatographic conditions as those demonstrated above and collect the effluent corresponding to that of the whole rhG-CSF peak, the sample solution (400 uL) containing the denatured/ reduced rhG-csf in 8 mol/L urea was diluted with the collected effluent and then the solution was left for 24 h at 4 degree. Procedures for the refolding and purification of rhG-CSF by IEC. Chromatographic runs were carried out at room temperature using a IEC column (20 × 1.2 cm i.d.) packed with Q Sepharose FF media with a bed volume of about 20 mL and connected to an ÄKTA Explorer 100A chromatographic system. The column was equilibrated with solution A consisting of 0.05 mol/L Tris, pH 8.0, 3.0 mol/L urea,1.0 mmol/L EDTA, 2.5 mmol/L GSH and 0.8 mmol/L GSSG. The sample solution (400 μL) containing the solubilized and denatured rhG-CSF with 2.8 mg/mL total protein (containing 1.15 mg/mL of rhG-CSF) in 8.0 mol/L urea solution was directly injected into the column. After washing the column with 30 mL of solution A, the refolding with simultaneous purification of rhG-CSF was accomplished after a linear gradient elution from 100% A to 60% B (solution B consistedof solution A plus 1.0 mol/L NaCl) in 90 mL at a flow rate of 5.0 mL/min with detection at 280 nm. The column was regenerated using 20 mL of 100% B.
Refolding Assay	SDS-PAGE
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	NOTE - protein was refolded into a buffer gradient from 0 to 60% buffer B. See above protocol.

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