Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cytotoxic T-lymphocyte protein 4 |
| Abbreviated Name | CLTA-4 |
| SCOP Family | V set domains (antibody variable domain-like) |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P16410 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Extracellular domain aa.1-121 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 122 |
| Molecular Weight | 13116.1 |
| Pi | 4.71 |
| Molecular Weight | 13116.1 |
| Disulphides | 1 |
| Full Sequence |
MKAMHV AQPAVVLASS RGIASFVCEY ASPGKATEVR VTVLRQADSQ VTEVCAATYM MGNELTFLDD SICTGTSSGN QVNLTIQGLR AMDTGLYICK VELMYPPPYY LGIGNGTQIY VIDPEP
|
| Notes | CTLA-4 can form a dimer, but in this study, refolding of the monomeric form was reported. See Cox et al, record no.1516 for refolding of dimeric form. |
| 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 | None |
| 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 | |
| Purity | |
| Notes | - Same protocol as for T lymphocyte activation antigen CD86/B7-2, record no.1502 |