Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Link module from human TSG-6
Abbreviated Name	Llink module
SCOP Family	Link domain
Structure Notes
Organism	Human
UniProt Accession	P98066
SCOP Unique ID	n/a
Structure Solved
Class	Alpha+Beta
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	98
Molecular Weight	10926.5
Pi	9.48
Molecular Weight	10926.5
Disulphides	2
Full Sequence	GVYHREARSGKYKLTYAEAKAVCEFEGGHLATYKQLEAARKIGFHVCAAGWMAKGRVGYPIVKPGPNCGFGKTGIIDYGIRLNRSERWDAYCYNPHAK
Notes	n/a

Expression
Report	Kahmann JD, Koruth R, Day AJ. (1997) Protein Expression and Purification, 9, 315-318
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	4 h
Expression Vector	pRK172
Expression Protocol	Protein expression. The Link module from human TSG-6, corresponding to amino acids 36 – 133 in the preprotein (5), was overexpressed in E. coli using the T7-vector, pRK172, as described previously (1). Briefly, starter cultures of BL21(DE3)pLysS, containing the VII-6-1mut8-5 plasmid (1), were grown in LB medium (10 g Bactotryptone, 5 g Oxoid yeast extract, 10 g NaCl/liter), containing 100 mg/ml ampicillin and 34 mg/ml chloramphenicol, at 37􏰈C, to give an OD600nm of 0.1. Cells were harvested by centrifugation (1000g for 10 min) and inoculated into ZB medium (10 g N-Z-amine A, 5 g NaCl/liter), containing antibiotics as above, to give 3 1*10 9 cells/litre. Cultures were grown in 2.5 liter baffled flasks (500 ml/flask) in a shaking incubator (150 rpm) at 37C. Heterologous protein expression was induced by addition of IPTG (0.1 mM final concentration) when the OD600nm was 0.4. Cells were harvested 4 h after induction by centrifugation for 10 min at 5000g and stored at -20􏰈C in 100 mM Tris-HCl, 5 mM EDTA, pH 8.0.
Method of Induction	IPTG
Cell Density at Induction	OD 0.4 = 600
Cell Disruption Method	Not stated
Lytic Agent	None
Pre-Refolding Purification	Size-exclusion chromatography
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	n/a
Solubilization Buffer	8 M guanidine-HCl, 50 mM Tris, pH 8.0, 100 mM
Refolding Buffer	50 mM ammonium acetate, pH 6.0, B-mercaptoethanol (12.8 ul)
Pre-Refolding Purification	Size-exclusion chromatography
Tag Cleaved	no tag
Refolding pH	6.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	48 h
Redox Agent	Beta-mercaptoethanol
Redox Agent Concentration	12.8 ul,12.8 ul
Refolding Protocol	Refolding of the Link module under nondenaturing conditions. The method described below is based on side the work of Haber and Anfinsen (8) on the rearrangement of disulfides in ribonuclease. The Link module preparation was resuspended in 50 mM ammonium acetate, pH 6.0, to a concentration of 500 mg/ml (45.78 uM, 40 ml final volume). A 100-fold molar excess of B-mercaptoethanol (12.8 ml) was added and this solution was incubated at 25􏰈C for 2 days, without stirring, in a 50-ml polypropylene centrifuge tube with holes in the lid for aeration. Protein was analyzed before and after this refolding procedure by HPLC on a Shandon 150 10-mm Hypersil C3-PEP (300 A˚ , 5 mm) column equilibrated in 80% (v/v) solvent A, 20% (v/v) solvent B at a flow rate of 3 ml/min. After injection of the sample initial conditions were maintained for 5 min followed by linear gradients of 20 – 35, 35 – 50, and 50 – 95% B over 10, 20, and 5 min, respectively. The A220nm of the eluent was monitored continuously.
Refolding Assay	HPLC
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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