Refolding Record:

Protein See all refolding records for this protein...
Protein Name	CD1a
Abbreviated Name	CD1a
SCOP Family	MHC antigen-recognition domain
Structure Notes
Organism	Human
UniProt Accession	P06126
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	284
Molecular Weight	32465.5
Pi	6.08425
Molecular Weight	32465.5
Disulphides	2
Full Sequence	MNPDG LKEPLSFHVI WIASFYNHSW KQNLVSGWLS DLQTHTWDSN SSTIVFLWPW SRGNFSNEEW KELETLFRIR TIRSFEGIRR YAHELQFEYP FEIQVTGGCE LHSGKVSGSF LQLAYQGSDF VSFQNNSWLP YPVAGNMAKH FCKVLNQNQH ENDITHNLLS DTCPRFILGL LDAGKAHLQR QVKPEAWLSH GPSPGPGHLQ LVCHVSGFYP KPVWVMWMRG EQEQQGTQRG DILPSADGTW YLRATLEVAA GEAADLSCRV KHSSLEGQDI VLYWEHHGS
Notes	n/a

Expression
Report	Altamirano MM, Woolfson A, Donda A, Shamshiev A, Briseno-Roa L, Foster NW, Veprintsev DB, De Libero G, Fersht AR, Milstein C. (2001) Proc. Natl. Acad. Sci. USA, 98, 3288-3293
Project Aim	Functional Studies
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21
Expression Temp	37.0
Expression Time	3h
Expression Vector	pET23d
Expression Protocol	Cells were grown at 37degC and expression was induced with 1mM IPTG for 3h.
Method of Induction	IPTG
Cell Density at Induction	OD =
Cell Disruption Method	French Press
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Column refolding: Oxidative refolding chromatography
Wash Buffer	10mM Tris, 1mM EDTA, pH 8.0
Solubilization Buffer	6M GdnHCl, 100mM potassium phosphate pH 8.0
Refolding Buffer	100mM potassium phosphate, 0.3M L-arginineHCl, 8mM oxidized glutathione, 0.1mM EDTA, 0.1M PMSF, pH 8.0
Pre-Refolding Purification	None
Tag Cleaved	no tag
Refolding pH	8.0
Refolding Temperature	25.0
Protein Concentration
Refolding Time	6min-12h
Redox Agent	GSSG
Redox Agent Concentration	8mM,8mM
Refolding Protocol	Cells were pelleted and lysed by French press. After centrifugation, inclusions bodies were washed several times in wash buffer containing 50 microgram/ml PMSF, then washed once in 1.0M urea before being flash frozen and stored at -70degC. Inclusion bodies were solubilised in solubilization buffer and reduced with 0.1M DTT. A refolding matrix was previously prepared comprising agarose-gel bead immobilised prokaryotic miniGroEL (apical domain of GroEL), DsbA (a protein disulphide isomerase) and a peptidyl0prolyl cis-trans isomerase(PPI). freshly denatured and reduced CD1 heavy chains were mixed with denatured and reduced beta-2-microglobulin light chains in various molar ratios (1:1 to 1:10, ideal 1:3) immediately before refolding. The mixture of heavy and light chains was then slowly added, mixed and diluted (1:100) into an aqueous suspention of the prepared refolding matrix. The mixtrue was rotated at 4degC for variou times (6min-12h) and centrifuged at <1000g for 5 min. The soluble fraction was retained and concentrated.
Refolding Assay	Immunoassay
Refolding Chaperones	None,PPI,DsbA,GroEL
Refolding Additives	None,L-Arginine
Additives Concentration	0.3M
Refolding Yield	>87%
Purity
Notes	- same refolding protocol for CD1b (record no. 1469) - refolding performed in absence and presence of synthetic ligand (ceramide galactoside 3-sulfate), stable complex formed with beta-2-microglobulin light chain only when refold in presence of ligand

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