Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Conglutinin
Abbreviated Name	n/a
SCOP Family	Unknown
Structure Notes
Organism	Bovine
UniProt Accession	P23805
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	352
Molecular Weight	35748.7
Pi	5.63
Molecular Weight	35748.7
Disulphides	2
Full Sequence	AEMTTFSQKILANACTLVMCSPLESGLPGHDGQDGRECPHGEKGDPGSPGPAGRAGRPGWVGPIGPKGDNGFVGEPGPKGDTGPRGPPGMPGPAGREGPSGKQGSMGPPGTPGPKGETGPKGGVGAPGIQGFPGPSGLKGEKGAPGETGAPGRAGVTGPSGAIGPQGPSGARGPPGLKGDRGDPGETGAKGESGLAEVNALKQRVTILDGHLRRFQNAFSQYKKAVLFPDGQAVGEKIFKTAGAVKSYSDAEQLCREAKGQLASPRSSAENEAVTQMVRAQEKNAYLSMNDISTEGRFTYPTGEILVYSNWADGEPNNSDEGQPENCVEIFPDGKWNDVPCSKQLLVICEF
Notes	n/a

Expression
Report	Lim BL, Holmskov U. (1996) Biochemical and Biophysical Research Com, 218, 260-6
Project Aim	Expression and identification
Fusion	N-terminal hexahistidine tag
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)
Expression Temp	37.0
Expression Time	2 h
Expression Vector	pGex-2T
Expression Protocol	Overexpression of the recombinant conglutinin in E. coli. A colony carrying the expression plasmid was inoculated into 20 ml of LA (Luria-Bertani medium + 100 mg/L ampicillin) and grown for 5 hours at 37°C. After keeping the bacterial culture at 4°C overnight, the cells were sedimented by centrifugation and inoculated into 1 litre LA. After 2.5 hours at 37°C, protein expression was induced by 0.1 mM IPTG and the culture was allowed to grow for 2 hours at 37°C. The cells were harvested by centrifugation at 5000g for 15 minutes. The cells were resuspended by E. coli lysis buffer (15ml/litre culture) and lysed by sonication (3 × 1 min).
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	Metal affinity chromatography
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	TBS (20 mM Tris-HCl, 140 mM NaCl, 0.05% (w/v) NaN3, pH 7.4)
Solubilization Buffer	1 × TBS, 6M Urea, 0.05% (v/v) Tween-20, 20 mM CaCl2
Refolding Buffer	TBS/NTC containing 20 mM CaCl, in a two step manner
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	no
Refolding pH	0.0
Refolding Temperature	4.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	The pellet, after centrifugation at 12000g for 15 minutes, contained the recombinant product. After washing twice in TBS, the pellet was dissolved in denaturing buffer, by gentle shaking at room temperature. Undissolved particulates were removed by centrifugation at 12000g and the supernatant was passed through a His-Band affinity column (3 cm × 10 mm) which had been equilibrated in the denaturing buffer. Non-binding proteins were washed away with the denaturing buffer and the fusion protein was eluted by elution buffer. For refolding, the protein was diluted 6 fold (final urea concentration 􏰰 1M) and 2 fold (final urea concentration 􏰰 0.5M) by TBS/NTC containing 20 mM CaCl2, in a two step manner. Each step was carried out overnight at 4°C. The refolded material was then dialysed against 3 changes of one litre TBS/NTC before passing through a N-acetylglucosamine-TSK column (16 mm × 150 mm). N-acetylglucosamine was coupled to TSK beads as described (13). After washing with TBS/NTC, the bound protein was eluted by TBS/EDTA.
Refolding Assay	ELISA
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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