Refolding Record:
| Protein | |
|---|---|
| Protein Name | T-cell receptor |
| Abbreviated Name | TCR |
| SCOP Family | V set domains (antibody variable domain-like); C1 set domains (antibody constant domain-like) |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01848 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Heterodimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 144 |
| Molecular Weight | 15927.8 |
| Pi | 4.57735 |
| Molecular Weight | 15927.8 |
| Disulphides | 4 |
| Full Sequence |
PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKS
NSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIG
FRILLLKVAGFNLLMTLRLWSS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Clements, Craig S., Kjer-Nielsen, Lars, MacDonald, Whitney A., Brooks, Andrew G., Purcell, Anthony W., McCluskey, James, Rossjohn, Jamie (2002) Acta Crystallographica Section D, 58, 2131-2134 |
| 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 |
| Expression Temp | 37.0 |
| Expression Time | 5 hrs |
| Expression Vector | PET30 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication/French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM Tris-HCl pH8.0, 1% TritonX-100, 1%deoxycholic acid, 100mM NaCl |
| Solubilization Buffer | 8M urea, 20mM Tris pH8.0, 0.5mM NaEDTA, 1mM DTT |
| Refolding Buffer | 100mM Tris-HCl pH8.0, 2mM NaEDTA, 400mM L-Arginine-HCl, 0.5mM Oxidised Glutathione, 5.0mM Reduced Glutathione |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 4uM final |
| Refolding Time | |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | 20 mg of alpha chain and 15 mg of beta chain are reduced with a further 1mM DTT and mixed. The protein is injected into cold, stirring refold buffer using a 27G needle. This is repeated at 12 and 24 hrs. At 36 hrs the protein is dialysed for 12 hrs against 15 volumes of 10mM Tris pH8.0, 100mM urea and then for a further 12 hrs against 10mM Tris pH8.0 |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 15 mg/L refold |
| Purity | |
| Notes | |