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 |
| 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 |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 8h |
| Redox Agent | DTT |
| Redox Agent Concentration | 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 step-wise dialysis with a sequential dialysis over 8h in 4, 2, 1, 0.1 and 0M urea in refolding buffer. 250mM L-arginine was added to the 1M urea buffer. The protein was refolded in the absence and presence of its binding partner protein, XPF(ERCC1-binding domain only) - it was found XPF was required for successful refolding. |
| 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 |