Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Endostatin
Abbreviated Name	EN
SCOP Family	Endostatin
Structure Notes
Organism	Mouse
UniProt Accession	P39061
SCOP Unique ID	n/a
Structure Solved
Class	Alpha+Beta
Molecularity	Monomer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	184
Molecular Weight	20376.0
Pi	9.09
Molecular Weight	20376.0
Disulphides	2
Full Sequence	HTHQDFQPVLHLVALNTPLSGGMRGIRGADFQCFQQARAVGLSGTFRAFLSSRLQDLYSIVRRADRGSVPIVNLKDEVLSPSWDSLFSGSQGQLQPGARIFSFDGRDVLRHPAWPQKSVWHGSDPSGRRLMESYCETWRTETTGATGQASSLLSGRLLEQKAASCHNSYIVLCIENSFMTSFSK
Notes	n/a

Expression
Report	Chura-Chambi RM, Genova LA, Affonso R, Morganti L. (2008) Analytical Biochemistry, 1, Epub ahead of p
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	BL21(DE3)pLysS
Expression Temp	37.0
Expression Time	16-h
Expression Vector	pET28
Expression Protocol	BL-21(DE3)pLysS was transformed with vector pET-28 containing the DNA sequence coding for the amino acids methionine, alanine, and six histidine residues at the N terminus followed by the sequence of murine ES. For ES expression, a colony was randomly picked from the transformants grown in Kan + LB plates (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 50 mg/L kanamycin) and inoculated into 15 mL of LB medium containing kanamycin. The 15 mL LB overnight culture (37 °C, 280 rpm) was diluted to a final volume of 500 mL of rich culture medium (2 × HKSII) [26], in 1000 mL shaker flasks. Expression of ES was induced with isopropyl-β-D-thiogalactopyranoside (0.5 mM) when the A600 nm reached 3.0. After incubating in the shaker for a 16-h period (37 °C, 280 rpm), bacteria were collected by centrifugation at 2500g for 10 min at 4 °C. The pellet was resuspended in 50 mL of 0.1 M Tris–HCl, pH 7.5, and 5 mM EDTA; lysozyme at a final concentration of 50 μg/mL was added; and the suspension was incubated at room temperature. The suspension was sonicated in the presence of 0.1% sodium deoxycholate, then centrifuged at 8000g for 10 min.
Method of Induction	IPTG
Cell Density at Induction	OD 3 = 600
Cell Disruption Method	Sonication
Lytic Agent	Lysozyme
Pre-Refolding Purification	not specified
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	0.05 M Tris–HCl, pH 7.5
Solubilization Buffer	0.05 M Tris–HCl, pH 7.5, 100 mM NaCl, 3 M urea
Refolding Buffer	50 mM Tris–HCl pH 7.5, 1 mM EDTA) containing the final concentrations of GdnHCl and oxidized (GSSG) and reduced (GSH) glutathione
Pre-Refolding Purification	not specified
Tag Cleaved	no tag
Refolding pH	7.5
Refolding Temperature	0.0
Protein Concentration	n/a
Refolding Time	16 h
Redox Agent	GSH/GSSG
Redox Agent Concentration	n/a
Refolding Protocol	Refolding buffer and sample pressurization The IBs were diluted in refolding buffer (50 mM Tris–HCl pH 7.5, 1 mM EDTA) containing the final concentrations of GdnHCl and oxidized (GSSG) and reduced (GSH) glutathione as indicated in the figures. Samples of 0.5 ml of the suspension were placed into plastic bags, which were vacuum/heat-sealed. The bags were placed in a pressure vessel, with a mixture of water and oil as pressure-transmitting fluid, and high pressure was applied (200 MPa, 2000 bar, or 29,000 psi) for 16 h. Samples were then centrifuged at 12000g for 15 min to remove remaining insoluble aggregates. The supernatant was dialyzed against 50 mM Tris–HCl, pH 7.5, buffer and centrifuged at 12,000g for 15 min to remove insoluble aggregates eventually formed during the dialysis process.
Refolding Assay	Unspecified
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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