Refolding Record:
| Protein | |
|---|---|
| Protein Name | Extracellular Superoxide Dismutase |
| Abbreviated Name | EC-SOD |
| SCOP Family | Cu,Zn superoxide dismutase-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P08294 |
| 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 | 243 |
| Molecular Weight | 26457.3 |
| Pi | 6.75481 |
| Molecular Weight | 26457.3 |
| Disulphides | 1 |
| Full Sequence |
MGSSHHHHHHSSGLVPRGSHM WT GEDSAEPNSD SAEWIRDMYA KVTEIWQEVM QRRDDDGTLH AACQVQPSAT LDAAQPRVTG VVLFRQLAPR AKLDAFFALE GFPTEPNSSS RAIHVHQFGD LSQGCESTGP HYNPLAVPHP QHPGDFGNFA VRDGSLWRYR AGLAASLAGP HSIVGRAVVV HAGEDDLGRG GNQASVENGN AGRRLACCVV GVCGPGLWER QAREHSERKK RRRESECKAA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | He HJ, Yuan QS, Yang GZ, Wu XF. (2002) Protein Expression and Purification, 24, 13-17 |
| Project Aim | Undefined |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 32.0 |
| Expression Time | 3-5h |
| Expression Vector | pET28a |
| Expression Protocol | An overnight culture of cells was diluted 1:100 in LB media and grown at 32degC until A600 reached 0.5. Expression was induced with 1mM IPTG and cells were grown a further 3-5h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | n/a |
| Solubilization Buffer | 20mM Tris-HCl, 1mM PMSF, 0.5M NaCl, 5mM imidazole, 6M urea pH 8.0 |
| Refolding Buffer | PBS, 150mM NaCl, 0.6M urea, 2mM GSH, 0.2mM GSSG, 0.5M arginine |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.4 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 48 |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2mM/0.2mM |
| Refolding Protocol | Refolding was initiated by 10-fold dilution into refolding buffer and carried out with constant slow stirring. The protein was then dialysed |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 0.5M |
| Refolding Yield | |
| Purity | |
| Notes | |