Refolding Record:

Protein See all refolding records for this protein...
Protein Name	granulocyte-macrophage colony-stimulating factor
Abbreviated Name	GM-CSF
SCOP Family	Short Chain Cytokines
Structure Notes
Organism	Human
UniProt Accession	P04141
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	128
Molecular Weight	14477.5
Pi	5.21
Molecular Weight	14477.5
Disulphides	Unknown
Full Sequence	APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITF ESFKENLKDFLLVIPFDCWEPVQE
Notes	n/a

Expression
Report	Doherty DH, Rosendahl MS, Smith DJ, Hughes JM, Chlipala EA, Cox GN. (2005) Bioconjug Chem, 16, 1291-1298
Project Aim	Undefined
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	W3110
Expression Temp	0.0
Expression Time	16 h
Expression Vector	pBBT268
Expression Protocol	Plasmids encoding wild type GM-CSF and GM-CSF cysteine analogs were transformed into E. coli strain W3110. An overnight culture of each strain was inoculated at an optical density at 600 nm of ~ 0.05 in 400 mL of Luria Broth media containing 10 μg/mL tetracycline. When the culture reached an optical density of ~ 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM and the induced culture was incubated overnight for about 16 h. The cells were pelleted by centrifugation and stored at −80°C. The cell pellet was thawed and treated with 5 mL of B-PER™ bacterial protein extraction reagent (Pierce Chemical Company, Rockford, IL) according to the manufacturer’s protocols.Insoluble material was recovered by centrifugation and resuspended in B-PER. This mixture was treated with lysozyme (200 μg/mL) for 10 min to further disrupt the cell walls, and MgCl2 (10 mM final concentration) and protease-free DNAse (2 μg/mL) were added. Insoluble GM-CSF was collected by centrifugation, resuspended in water and recentrifuged.
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	None
Lytic Agent	Lysozyme
Pre-Refolding Purification	not specified
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	n/a
Solubilization Buffer	10 mL of 8 M urea, 25 mM cysteine, 20 mM Tris Base
Refolding Buffer	100 mL of 20 mM Tris, 40 μM copper sulfate, 15% glycerol, pH 8.0
Pre-Refolding Purification	not specified
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	The insoluble pellet was dissolved in 10 mL of 8 M urea, 25 mM cysteine, 20 mM Tris Base, stirred for 30 min at room temperature and diluted into 100 mL of 20 mM Tris, 40 μM copper sulfate, 15% glycerol, pH 8.0. The refold mixture was held at 4°C for 2 days, centrifuged and loaded onto a 5 mL Q-Sepharose HiTrap column (Amersham Biosciences Corporation, Piscataway, NJ) equilibrated in 20 mM Tris, pH 8.0 (Buffer A). The bound proteins were eluted with a linear salt gradient from 0–35% Buffer B (1M NaCl, 20 mM Tris, pH 8). Column fractions were analyzed by non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fractions containing primarily GM-CSF were pooled. The Q-Sepharose pool was diluted with an equal volume of 30% ammonium sulfate and loaded onto a 1 mL Phenyl HP HiTrap column (Amersham Biosciences Corporation) previously equilibrated with 15% ammonium sulfate in 20 mM sodium phosphate, pH 7.5. GM-CSF was recovered from the column by elution with a reverse salt gradient (15% ammonium sulfate to 0% ammonium sulfate in 20 mM sodium phosphate, pH 7.5). Column fractions were analyzed by non-reducing SDS-PAGE and fractions containing GM-CSF and no detectable contaminants were pooled.
Refolding Assay	SDS-PAGE
Refolding Chaperones	None
Refolding Additives	Glycerol
Additives Concentration	15%
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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