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 | Dimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Extracellular domain aa.1-125 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 125 |
| Molecular Weight | 13374.2 |
| Pi | 4.38573 |
| Molecular Weight | 13374.2 |
| Disulphides | 1 |
| Full Sequence |
AMHV AQPAVVLASS RGIASFVCEY ASPGKATEVR VTVLRQADSQ VTEVCAATYM MGNELTFLDD SICTGTSSGN QVNLTIQGLR AMDTGLYICK VELMYPPPYY LGIGNGTQIY VIDPEPCPDS D
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Cox GN, Pratt D, Smith D, McDermott MJ, Vanderslice RW (1999) Protein Expression and Purification, 17, 26-32 |
| Project Aim | Drug Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | HMS/DE3 |
| Expression Temp | 37.0 |
| Expression Time | 6h |
| Expression Vector | pT5T |
| Expression Protocol | Cells were grown in complex medium at 37degC until OD660 reached 10. Protein expression was induced by the addition of 1mM IPTG with incubation for a further 6h. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 25mM NaCl, 50mM TrisHCl pH 7.5, 1mM DTT |
| Solubilization Buffer | 6M GdnHCl, 50mM Tris pH 8.5 |
| Refolding Buffer | 50mM Tris pH 9.5, 0.6M GdnHCl, 0.3mM DTT |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 9.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 3days |
| Redox Agent | DTT |
| Redox Agent Concentration | 0.3mM |
| Refolding Protocol | Cells were harvested by centrifugation and resuspended at 400g/2L in wash buffer and passed three times through a Rannie minimill at 10000psi. The cell lysate was centrifuged and the inclusion bodies were washed several times in wash buffer. The pellet was then suspended in wash buffer and homogenized twice using a Polytron 3000mixer set at 8000rpm. The inclusion bodies were then collected by centrifugation and suspended in solubilization buffer at a rate of 30g inclusion bodies in 600ml buffer using a Polytron PT 3000 mixer. DTT was added to a final concentration of 6mM and the suspension was stirred slowly at room temperature for 60min. The solution was then centrifuged (30min x 17700g)and the supernatant was added slowly to 11.4 L of refolding buffer. The solution was incubated for 3 days at 4degC and then centrifuged to remove insoluble matierial. The supernatant was concentrated to 1-2L and centrifuged and filtered before being loaded onto a 50ml Resource Q column previously equilibrated with 20mM Tris pH 7 (Buffer A). The protein was eluted in 15 column volumes (750ml) using a linear gradient to 60% 1M NaCl, 20mM Tris pH 8.0 (Buffer B). Following non-reducing SDS-PAGE analysis, fractions containing dimers were loaded onto a S100 gel filtration column in 20mM sodium acetate, 250mM NaCl, pH 5.5. |
| Refolding Assay | HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |