Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Lysozyme
Abbreviated Name	Lysozyme
SCOP Family	C-type Lysozyme
Structure Notes
Organism	Human
UniProt Accession	P61626
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	130
Molecular Weight	14700.7
Pi	9.27
Molecular Weight	14700.7
Disulphides	4
Full Sequence	KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV
Notes	n/a

Expression
Report	Li M, Su Z (2002) Chromatographia, 56, 33-38
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
Expression Temp	28.0
Expression Time	7 h
Expression Vector	pBV-HLy
Expression Protocol	E. Coli BL21 was the host cell for plasmid pBV-Hly (a gift plasmid from Prof. N.J. Qian, Institute of Microbiology, Chinese Academy of Sciences), which was cultured in RM culture medium at 28 c for 4 h [20]. Product formation was induced by temperature shift from 28 to 42 c and in- cubation for 3 h. The culture was centrifuged at 6000 g and 4 c for 20 min (Centrifuge 3K30, Martin Christ GmbH, Germany). The precipitate was re-suspended using buffer (0.1 mol L 1 Tris-HC1 at pH 8.0) and centrifuged again. The precipitated microorganism was re-suspended to a concentration of 100 g L 1 and disrupted by ultrasonication (Sonics and Materials Inc, USA, 500W) for 4 * 8 min bursts. The homogenate was centrifuged at 12000 g and 4 c for 15 min. The insoluble cell fractions (the inclusion body and cell debris) were washed for three times with 0.1 mol U 1 Tris-HC1 at pH 8.0 containing 0.5 mmol L 1 EDTA and 0.5% (v/v) Triton X-100. The remaining pellet containing the inclusion body was stored at -20 c.
Method of Induction	Temperature Shift
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	ultrasonication
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	0.1 mol U 1 Tris-HC1 at pH 8.0 containing 0.5 mmol L 1 EDTA and 0.5% (v/v) Triton X-100
Solubilization Buffer	8 moll 1 urea, 0.05moll 1 Tris-HC1 (pH 8.0), and 0.1 mol L 1 dithiothreitol (DTT).
Refolding Buffer	0.1 mol L 1 Tris-HC1 at pH 8.0 containing 0.1 mol L 1 (NH4)2SO4, 1 mol L 1 urea, 3 mmol L 1 reduced glutathione (GSH) and 0.3 mmol L 1 oxidized glutathione (GSSG)
Pre-Refolding Purification	None
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	8 h
Redox Agent	GSH/GSSG
Redox Agent Concentration	3/0.3 mM,3/0.3 mM,3/0.3 mM
Refolding Protocol	Dilution was performed by direct addition of the protein solution into refolding buffer (0.1 mol L 1 Tris-HC1 at pH 8.0 containing 0.1 mol L 1 (NH4)2SO4, 1 mol L 1 urea, 3 mmol L 1 reduced glutathione (GSH) and 0.3 mmol L 1 oxidized glutathione (GSSG)) at varying final protein concentration.
Refolding Assay	Protein activity assay
Refolding Chaperones	None
Refolding Additives	(NH4)2SO4
Additives Concentration	0.1 mol/l
Refolding Yield	35%
Purity
Notes	n/a

Back to refolding records...