Refolding Record:
| Protein | |
|---|---|
| Protein Name | P53 tumor suppressor |
| Abbreviated Name | p53 |
| SCOP Family | p53 DNA-binding domain-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P04637 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Tetramer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 393 |
| Molecular Weight | 43653.2 |
| Pi | 6.33163 |
| Molecular Weight | 43653.2 |
| Disulphides | 0 |
| Full Sequence |
MEEPQSDPSV EPPLSQETFS DLWKLLPENN VLSPLPSQAM DDLMLSPDDI EQWFTEDPGP DEAPRMPEAA PPVAPAPAAP TPAAPAPAPS WPLSSSVPSQ KTYQGSYGFR LGFLHSGTAK SVTCTYSPAL NKMFCQLAKT CPVQLWVDST PPPGTRVRAM AIYKQSQHMT EVVRRCPHHE RCSDSDGLAP PQHLIRVEGN
LRVEYLDDRN TFRHSVVVPY EPPEVGSDCT TIHYNYMCNS SCMGGMNRRP ILTIITLEDS SGNLLGRNSF EVRVCACPGR DRRTEEENLR KKGEPHHELP PGSTKRALPN NTSSSPQPKK KPLDGEYFTL QIRGRERFEM FRELNEALEL KDAQAGKEPG GSRAHSSHLK SKKGQSTSRH KKLMFKTEGP DSD
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Bell S, Hansen S, Buchner J (2002) Biophysical Chemistry, 96, 243-257 |
| Project Aim | Structure-Function |
| 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 | 3.5h |
| Expression Vector | pET-3a |
| Expression Protocol | Cells were grown in Luria Broth (LB) medium with ampicillin (100 microg/ml) at 37degC and at a constant pH of 7.2. Recombinant protein expression was induced at an OD578 nm of 0.6 by addition of 1 mM IPTG. Three and a half hours after induction, cells were harvested by centrifugation and stored at –70degC. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 578 = 0.6 |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 0.1M Tris-HCl, 30mM EDTA, 3% Triton X-100, 0.8M NaCl pH 7 |
| Solubilization Buffer | 100mM Tris-HCl, 6M guanidinium chloride, 50mM DTT, pH 8 |
| Refolding Buffer | 50mM sodiuim diphosphate, 1M L-arginine, 2mM DTT, 0.2mM ZnCl2 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 15.0 |
| Protein Concentration | 75-100 micrograms/ml |
| Refolding Time | 10h |
| Redox Agent | DTT |
| Redox Agent Concentration | 2mM |
| Refolding Protocol | After solubilisation for 2h at room temperature, the pH of the solubilisation buffer was shifted to 2. Refolding was initiated by 100-150 fold dilution in refolding buffer with incubation at 15C for 10h prior to dialysis overnight against 50mM sodium diphosphate, 4mM DTT, 5% glycerol pH 8 at 4C. |
| Refolding Assay | DNA binding |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 1M |
| Refolding Yield | |
| Purity | |
| Notes | |