Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Monoclonal anti-idiotypic Schistosoma japonicum antibody NP30 immunoglobulin lig
Abbreviated Name	MAb -LIGHT
SCOP Family	Unknown
Structure Notes
Organism	Mouse
UniProt Accession	Q9U410
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	106
Molecular Weight	11477.8
Pi	6.43
Molecular Weight	11477.8
Disulphides	Unknown
Full Sequence	ENLLTQSPAIMSASPGEKVTMTCSASSSVSYVYWYLQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWTSYPFTFGSGTKLELK
Notes	n/a

Expression
Report	Fan K, Zhou Q, Wang H, Guo Y. (2005) Hybridoma (Larchmt)., 24, 309-313
Project Aim	Purification & characterization
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	4 h
Expression Vector	pET32a
Expression Protocol	Expression, purification, and refolding of fusion protein his-tagged LIGHT The DNA fragment containing the mouse LIGHT extracellular domain was amplified by PCR using the primers 5\'- aggtaccgatgacgacgacaagagactgcatcaacgtctt-3 \'(sense) and 5\'- aggatcctcagaccatgaaagctccgaa-3\' (antisense) from the plasmid containing full-length LIGHT (gift of Professor Lieping Chen, National Institutes of Health, Bethesda, MD). Polymerase chain reaction, (PCR) fragments were digested, retrieved and inserted into the BamHI-KpnI-digested pET-32a(+) vector. Re- combined plasmids were transformed into Escherichia coli BL21(DE3). A culture was grown in LB medium containing 100 ug/mL ampicillin at 37°C until the OD600was 0.6, at which point IPTG (1.0 mM, final) was added to induce protein expression. The bacteria was then harvested by centrifugation 4 h later.
Method of Induction	IPTG
Cell Density at Induction	OD 0.6 = 600
Cell Disruption Method	Sonication
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Column refolding: Nickel-chelating chromatography
Wash Buffer	20 mM Tris-HCl, 0.5 M NaCl, 2% Triton X-100, pH 8.0
Solubilization Buffer	8 M urea, 0.1 M NaH2PO4, 0.1 M Tris-HCl pH 8.0
Refolding Buffer	20 mL of 20 mM Tris- HCl, 0.5 M NaCl, 5mM imidazole, 8 M urea, pH 8.0
Pre-Refolding Purification	None
Tag Cleaved	no
Refolding pH	8.0
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	The bacteria was then harvested by centrifugation 4 h later. The cell pellet was resuspended in 20 mM Tris-HCl pH 8.0, and then disrupted with sonication on ice for 30 min and centrifuged at high speed for 20 min at 4°C. The pellet, containing the inclu- sion bodies, is resuspended in a cold buffer (2 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton X-100, pH 8.0), and soni- cated as above. The pellet material was washed once in buffer lacking urea and was resuspended in the denaturing buffer con- taing 8 M urea, 0.1 M NaH2PO4, 0.1 M Tris-HCl pH 8.0, and then stirred for 1–3 h at room temperature. The sample was then centrifuged at high speed for 30 min and filtered through a 0.45- 􏰏m filter, loaded in the Ni􏰑-loaded chelating 5-mL column, and then the column was washed with 20 mL of 20 mM Tris- HCl, 0.5 M NaCl, 5mM imidazole, 8 M urea, pH 8.0. Refold- ing of the bound protein is performed by the use of a linear 8–0 M urea gradient, starting with the wash buffer above and fin- ishing with one without urea. The refolded recombinant protein was eluted using a 30-mL linear gradient starting with 20 mM Tris-HCL, 0.5 M NaCl, 20 mM imidazole pH 8.0, and ending with the same buffer including 300 mM imidazole. Eluted so- lutions were estimated by coomassie staining after sodium do- decyl sulfate–polyacrylamide electrophoresis (SDS-PAGE). Purified fusion proteins were used as immunogen or screening antigen in enzyme-linked immunosorbent assay (ELISA) for the identification of MAb against LIGHT.
Refolding Assay	ELISA
Refolding Chaperones	None
Refolding Additives	None
Additives Concentration	n/a
Refolding Yield	n/a
Purity	n/a
Notes	n/a

Back to refolding records...