Refolding Record:
| Protein | |
|---|---|
| Protein Name | ERCC1 |
| Abbreviated Name | ERCC1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P07992 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | C-terminal XPF-binding domain (aa.224-297) |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 74 |
| Molecular Weight | 8261.6 |
| Pi | 6.35 |
| Molecular Weight | 8261.6 |
| Disulphides | 0 |
| Full Sequence |
MEKLEQD FVSRVTECLT TVKSVNKTDS QTLLTTFGSL EQLIAASRED LALCPGLGPQ KARRLFDVLH EPFLKVP
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Choi YJ, Ryu KS, Ko YM, Chae YK, Pelton JG, Wemmer DE, Choi BS (2005) J Biol Chem, 280, 28644-28652 |
| Project Aim | Structural Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pET21a |
| Expression Protocol | not stated |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution with complex partner subunits |
| Wash Buffer | n/a |
| Solubilization Buffer | 100mM sodium phosphate pH 8, 200mM NaCl, 1mM DTT, 6M urea |
| Refolding Buffer | 100mM sodium phosphate pH 8, 200mM NaCl, 1mM DTT, 10mM GSH, 1mM GSSG, 250mM L-arginine |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 20h |
| Redox Agent | GSH/GSSG/DTT |
| Redox Agent Concentration | 10mM/1mM/1mM |
| Refolding Protocol | Inclusion bodies were resuspended in solubilization buffer and purified using a Sephacryl S200 resin under denaturing conditions. The protein was then refolded by rapid dilution in the absence and presence of the partner protein XPF(ERCC1-binding domain only) - it was found that refolding was only successful in the presence of XPF. Refolding was performed for 20h with stirring, after which, the refolding solution was dialysed against 100mM sodium phosphate pH 8, 1mM DTT, 100mM NaCl |
| Refolding Assay | 15N -1H chemical shifts (ppm),Gel filtration chromatography,Complex Assembly,Ultracentrifugation equilibrium sedimentation |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 250mM |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |