Refold - Hepcidin

Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Hepcidin_20
Abbreviated Name	Hepc-20
SCOP Family	Hepcidin
Structure Notes
Organism	Human
UniProt Accession	P81172
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Unknown

Expression
Report	Gerardi G, Biasiotto G, Santambrogio P, Zanella I, Ingrassia R, Corrado M, Cavadini P, Derosas M, Levi S, Arosio P. (2005) Blood Cells Mol Dis., 35, 177-81
Project Aim	Recombinant Protein Expression
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21 pLysS
Expression Temp	0.0
Expression Time	00
Expression Vector	pET12a
Expression Protocol	Protein expression was induced in the transformed BL21pLys E. coli strain with 0.4 mM IPTG, as described in [19] and [20]. Purification of FTH-wt and FTH–Hep was performed as previously described for recombinant H ferritin [19] and [20].
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Not stated
Lytic Agent	None
Pre-Refolding Purification	not specified
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	n/a
Solubilization Buffer	8 M urea at pH 5
Refolding Buffer	10 mM DTT were diluted or dialyzed in 0.05 M phosphate buffer pH 7.4.
Pre-Refolding Purification	not specified
Tag Cleaved	no tag
Refolding pH	7.4
Refolding Temperature	0.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	DTT
Redox Agent Concentration	10 mM
Refolding Protocol	The insoluble GST–Hep and FTH–DE–Hep were resolubilized with 8 M urea at pH 5 yielding preparations >80% pure. In renaturation experiments the proteins added of 10 mM DTT were diluted or dialyzed in 0.05 M phosphate buffer pH 7.4. FTH-wt and FTH–Hep were made iron-free by incubation with 1% thioglycolic acid, pH 5.5, and 2,2-bipyridyle followed by extensive dialysis against 0.1 M HEPES, pH 7.0 [20]. To study the rate of iron oxidation the apoproteins (0.1 μM) in 20 mM HEPES pH 7.0 were added of 100 μM ferrous ammonium sulfate and the reaction monitored by the variation of absorbance at 310 nm [20]. To form the iron chromophore, the apoproteins at the concentration of 2.5 mg/ml in Tris–HCl 20 mM pH 7.4, 150 mM potassium acetate, 1.4 mM MgCl2, 70 mM DTT, 5% glycerol, were added of various concentrations of Na2S and ferrous ammonium sulfate and their UV/Vis spectra recorded. Iron concentration was determined after reduction and chelation [20].
Refolding Assay	Unspecified
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	Refolding temperature is not stated