Refolding Record:

Protein See all refolding records for this protein...
Protein Name	T lymphocyte activation antigen CD86/B7-antigen
Abbreviated Name	CD86/B2-7
SCOP Family	V set domains (antibody variable domain-like)
Structure Notes
Organism	Human
UniProt Accession	P42081
SCOP Unique ID	n/a
Structure Solved
Class	Beta
Molecularity	Monomer

Construct
Full Length	n
Domain	V-type Ig (receptor binding) domain, aa.1-109
Chimera	n/a
Variants	n/a
Chain Length	110
Molecular Weight	12833.6
Pi	6.49
Molecular Weight	12833.6
Disulphides	Unknown
Full Sequence	MLKIQA YFNETADLPC QFANSQNQSL SELVVFWQDQ ENLVLNEVYL GKEKFDSVHS KYMGRTSFDS DSWTLRLHNL QIKDKGLYQC IIHHKKPTGM IRIHQMNSEL SVLA
Notes	n/a

Expression
Report	Zhang X, Schwartz JCD, Almo SC, Nathenson SG (2002) Protein Expression and Purification, 25, 105-113
Project Aim	Crystallography
Fusion	None
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	3h
Expression Vector	pET3a
Expression Protocol	Cells were grown at 37degC in LB medium supplemented with 0.05mg/ml carbenicillin until OD600 reached 0.5-0.7, at which time protein expression was induced by the addition of 1mM IPTG. Incubation then continued for 3h, followed by harvesting of cells by centrifugation (20min x 5000g, 4degC).
Method of Induction	IPTG
Cell Density at Induction	OD 600 = 0.5-0.7
Cell Disruption Method	French Press
Lytic Agent	None
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	10mM TrisHCl, 100mM NaCl, 0.5% Triton X-100, 1mM EDTA, 10mM DTT, pH 8.0
Solubilization Buffer	10mM sodium acetate, 6M GdnHCl, 5mM EDTA, 1mM DTT pH 4.6
Refolding Buffer	100mM TrisHCl, 0.4M arginine HCl, 1mM EDTA, 5mM cysteamine, 0.5mM cystamine pH 8.5
Pre-Refolding Purification	None
Tag Cleaved	no tag
Refolding pH	8.5
Refolding Temperature	4.0
Protein Concentration
Refolding Time	8h
Redox Agent	cysteamine/cystamine
Redox Agent Concentration	5mM/0.5mM
Refolding Protocol	The cell pellet was resuspended in lysis buffer (~5ml per L bacterial culture) containing 10mM TrisHCl, 150mM NaCl, 25%(w/v) sucrose, 1mM EDTA, 10mM DTT, pH 8.0. Cells were lysed by 3 passes through a french pressure cell at 1000psi. Inclusion bodies were collected by centrifugation (15min x 10000g, 4degC) and then washed three times with wash buffer and one final time with wash buffer excluding Triton X-100. The inclusion bodies were then dissolved in solubilization buffer and centrifuged at 15000g. The supernatent was then filtered through 0.45microns and the protein concentration was determined by absorption at 280nm (extinction coefficient = 16500 /M/cm). 16mg of solubilized inclusion bodies were then diluted into 16ml of buffer containing solubilization buffer without DTT and then rapildy diluted over a few seconds into 1L of refolding buffer at 4degC with figorous stirring. Five more aliquots of protein (16mg each) were then added to the refolding mixture every 6-8h. The refolded protein was then passed down a Superdex G75 column in 10mM TrisHCl, 50mM NaCl pH 8.0. The protein was then buffer exchanged to 10mM TrisHCl, 20mM NaCl pH 8.5 using a 350ml Amicon concentration cell with a 3K MW cutoff membrane. The protein was then centrifuged (15000g) and filtered and then loaded onto a 1ml MonoQ column preequilibrated with 10mM Tris pH 8.0. The protein was eluted with a 0-500mM NaCl linear gradient spanning 20 column volumes. The protein was concentrated and the buffer was exchanged to 10mM TrisHCl. 20mM NaCl pH 8.0.
Refolding Assay	Gel filtration chromatography
Refolding Chaperones	None
Refolding Additives	None,L-Arginine
Additives Concentration	0.4M
Refolding Yield	35mg/100mg IBs
Purity
Notes	Same protocol for monomeric Cytotoxic T-lymphocyte protein 4 (CTLA-4), record no.1503

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