Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Macrophage colony-stimulating factor
Abbreviated Name	MCSF
SCOP Family	Short Chain Cytokines
Structure Notes
Organism	Human
UniProt Accession	P09603
SCOP Unique ID	n/a
Structure Solved
Class	Alpha
Molecularity	Dimer

Construct
Full Length	n
Domain	aa.1-223
Chimera	n/a
Variants	n/a
Chain Length	223
Molecular Weight	25134.4
Pi	4.84097
Molecular Weight	25134.4
Disulphides	6
Full Sequence	EEVSEYCS HMIGSGHLQS LQRLIDSQME TSCQITFEFV DQEQLKDPVC YLKKAFLLVQ DIMEDTMRFR DNTPNAIAIV QLQELSLRLK SCFTKDYEEH DKACVRTFYE TPLQLLEKVK NVFNETKNLL DKDWNIFSKN CNNSFAECSS QDVVTKPDCN CLYPKAIPSS DPASVSPHQP LAPSMAPVAG LTWEDSEGTE GSSLLPGEQP LHTVDPGSAK QRPPR
Notes	n/a

Expression
Report	Tran-Moseman A, Schauer N, De Bernardez Clark E (1999) Protein Expression and Purification, 16, 181-189
Project Aim	Structure-Function
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	GI724
Expression Temp	37.0
Expression Time	4h
Expression Vector	pALM3
Expression Protocol	Cells were grown in 10L fermentor medium (see paper for full details). The medium was adjusted to pH 7.2 and supplemented with 50mM glucose prior to inoculation. When the cell culture reached a density of 5g (dry cell weight)/L, protein expression was induced by the addition of a tryptophan-rich induction solution. The cells were incubated a further 4h then harvested.
Method of Induction	Tryptophan-rich induction solution
Cell Density at Induction	OD =
Cell Disruption Method	High pressure homogenization
Lytic Agent	None
Pre-Refolding Purification	HPLC
Solubility	soluble

Refolding
Refolding Method	Dilution
Wash Buffer	n/a
Solubilization Buffer	8M GdmCl, 50mM DTT, 10mM EDTA, 50mM Tris pH 8.0
Refolding Buffer	5mM EDTA, 50mM Tris pH 8.0, various concentrations of GSH/GSSG
Pre-Refolding Purification	HPLC
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	4.0
Protein Concentration
Refolding Time	64h
Redox Agent	GSH/GSSG
Redox Agent Concentration	various concentrations,various concentrations,various concentrations
Refolding Protocol	Cell pellets were resuspended in 50mM Tris, 10mM EDTA pH 8.0 at 100g cells/L. The cell suspension was homogenized and then centrifuged (8000g, 30min, 4degC). GdmCl and DTT were added to the inclusion bodiy suspension to yield solubilized protein in solubilization buffer. The suspension was incubated for 1.5h at room temperature and the pH was adjusted to 5 with acetic acid, followed by centrifugation (8000g, 30min, 4degC) and filtration. The protein was then purified on a preparative RP-HPLC Vydac C4 column. Purified fractions were freeze dried and stored at -80degC. The freeze-dried protein was resuspended in 8M urea, 5mM EDTA, 50mM Tris pH 8.0, 10mM DTT for about 45min at room temperature. The volume of the solution was adjusted to give a final protein concentration of 1-6mg/ml. The protein was then refolded by rapid dilution into refolding buffer. Refolding was performed over different time ranges, maximal refolding occured at 64h.
Refolding Assay	Bioactivity
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	NULL
Refolding Yield
Purity
Notes	Refolding also tried without pre-refolding purification, but was not as productive (see paper for more details) Refolding experiments also performed at 22degC (see paper for more details)

Back to refolding records...