Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Lysophospholipase
Abbreviated Name	n/a
SCOP Family	Unknown
Structure Notes
Organism	Pyrococcus furiosus
UniProt Accession	Q8U3I6
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Dimer

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	257
Molecular Weight	28772.3
Pi	7.2
Molecular Weight	28772.3
Disulphides	Unknown
Full Sequence	MTQVYKAKFGTPNRGWVIIVHGLGEHSGRYSKLVSMLVNEGYAVYTFDWPGHGKSPGKRGHTSVEEAMEIIDFIIEEINDKPFLFGHSLGGLTVIRYAETRPEKIRGVIASSPALAKSPKTPSFMVALAKILGVLLPSLTLSNGIDPNLLSRNPDAVKRYIEDPLVHDRISAKLGRSIFKNMDLAHREAHKIKVPVLLLVGTGDVITPPEGARKLYGEIKVEDKEIVEFEGAYHEIFEDPEWGEEFHKKIVEWIKKH
Notes	n/a

Expression
Report	Chandrayan SK, Dhaunta N, Guptasarma P. (2008) Protein Expression and Purification, 59, 327-33
Project Aim	Protein refolding
Fusion	N-terminal +C terminal hexahistidine tag
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	3 h
Expression Vector	pET23a
Expression Protocol	The PCR product was digested with SmaI and SalI enzymes, and first cloned into the multiple cloning site of the vector pGEX-KG (Amersham Biosciences) before it was sub- cloned between the BamHI and XhoI sites of the multiple cloning site of the vector pET23a (Novagen Inc.) for expression. The clone was transformed into E. coli BL21DE3 (pLysS) and the protein was expressed through induction with IPTG (1 mM) after approximately three hours of growth of cells at 37 °C (at an A600 of 0.6–0.8). Cells were harvested after overnight growth in the presence of IPTG.
Method of Induction	IPTG
Cell Density at Induction	OD 0.6-0.8 = 600
Cell Disruption Method	Not stated
Lytic Agent	None
Pre-Refolding Purification	Metal affinity chromatography
Solubility	partial

Refolding
Refolding Method	Dialysis
Wash Buffer	n/a
Solubilization Buffer	6 M guanidinium hydrochloride (Gdm HCl) containing 1 M NaCl in 50 mM Tris
Refolding Buffer	20 mM phosphate buffer pH 7
Pre-Refolding Purification	Metal affinity chromatography
Tag Cleaved	no
Refolding pH	7.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Cells were pelleted and resuspended in the presence of 6 M guanidinium hydrochloride (Gdm HCl) containing 1 M NaCl in 50 mM Tris, pH 7.0. After overnight incubation in denaturant, the resuspension was heated to 80 °C for 30 min, and centrifuged to pellet out the cell and membrane debris resulting from cell lysis. The GdmHCl-containing supernatant containing the lysophospholipase was loaded onto a 2.5 ml Ni-NTA affinity column (Sigma–Aldrich) using standard protocols. Following flow-through and washing steps, elution of bound lysophospholipase was carried out sequentially with 8 M urea-containing buffers of pH 5.9, and 4.5. Following elution, the urea was dialyzed out against 20 mM phosphate buffer of pH 7.0, to obtain a mixture of soluble protein and insoluble precipitated protein. The soluble protein was quantified through absorption measurements at 280 nm, using the extinction coefficient predicted for the protein, translating to a concentration of 0.97 mg/ ml for an OD of 1.0 at 280 nm.
Refolding Assay	Fluorescence,Activity assay,Circular Dichroism (uv-CD),Thermal melting analyses
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	0.23 mg/4 g
Purity	n/a
Notes	n/a

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