Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Mannose-binding lectin
Abbreviated Name	n/a
SCOP Family	alpha-D-mannose-specific plant lectins
Structure Notes
Organism	Galanthus nivalis (Common snowdrop)
UniProt Accession	P30617
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	109
Molecular Weight	12048.5
Pi	4.37
Molecular Weight	12048.5
Disulphides	Unknown
Full Sequence	SCLSDNILYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWATG
Notes	n/a

Expression
Report	Fitches E, Audsley N, Gatehouse JA, Edwards JP. (2002) Insect Biochem Mol Biol., 32, 1653-1661
Project Aim	Drug Studies
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	3 h
Expression Vector	PET14b
Expression Protocol	For protein overproduction, BL21(DE3)pLysS cells containing the GNA and FP expression constructs were cultivated with shaking at 37 °C in 1 L of LB broth containing 50 μg/ml carbenicillin and 34 μg/ml chloramphenicol. Cultures were induced to express the inserted genes with 0.4 mM isopropyl β-D- thiogalactoside when an O.D. of 0.6–0.7 had been attained. Cultivation at 37 °C was continued for 3 h post induction
Method of Induction	IPTG
Cell Density at Induction	OD 0.6-0.7 = 600
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	Metal affinity chromatography
Solubility	insoluble

Refolding
Refolding Method	Dilution/Dialysis combination
Wash Buffer	20 mM Tris, 0.5 M NaCl, 20 mM imidazole, 6 M urea, pH 7.8
Solubilization Buffer	20 mM Tris, 0.5 M NaCl, 0.3 M imidazole, 6 M urea, pH 7.8
Refolding Buffer	20 mM Tris, 1 M urea, pH 7.8 and dH2O (containing approx. 0.01% ammonium bicarbonate)
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	no
Refolding pH	7.8
Refolding Temperature	0.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Cells were harvested by centrifugation (30 min at 6000×g) and re-suspended in 100 ml binding buffer (20 mM Tris, 0.5 M NaCl, 5 mM imidazole, 6 M urea, pH 7.8). Cells were lysed by sonication and cellular debris removed by centrifugation (20 min at 10,000×g). Recombinant GNA and FP were purified by affinity chromatography using Ni-NTA Superflow resin (Qiagen). Columns (5 ml) loaded at 1 ml/min were washed with wash buffer (20 mM Tris, 0.5 M NaCl, 20 mM imidazole, 6 M urea, pH 7.8) and eluted with elution buffer (20 mM Tris, 0.5 M NaCl, 0.3 M imidazole, 6 M urea, pH 7.8). Purified GNA and FP fractions were diluted to 10–50 μg/ml in 20 mM Tris, 4 M urea, pH 7.8, and dialysed sequentially against 20 mM Tris, 1 M urea, pH 7.8 and dH2O (containing approx. 0.01% ammonium bicarbonate). After dialysis renatured proteins were filtered (0.45 μm), frozen in liquid nitrogen and freeze-dried.
Refolding Assay	Haemagglutination assays
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	The refolding temperature is not stated

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