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 | 223 |
| Molecular Weight | 24132.8 |
| Pi | 6.32 |
| Molecular Weight | 24132.8 |
| Disulphides | 1 |
| Full Sequence |
WTGEDSAEPNSDSAEWIRDMYAKVTEIWQEVMQRRDDDGALHAACQVQPSATLDAAQPRVTGVVLFRQLAPRAKLDAFFALEGFPTEPNSSSRAIHVHQFGDLSQGCESTGPHYNPLAVPHPQHPGDFGNFAVRDGSLWRYRAGLAASLAGPHSIVGRAVVVHAGEDDLGRGGNQASVENGNAGRRLACCVVGVCGPGLWERQAREHSERKKRRRESECKAA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Ryu K, Kim YH, Kim Y, Lee JS, Jeon B, Kim TY. (2008) J Microbiol Biotechnol., 18, 1648-54 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | E coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pET-EC-SOD |
| Expression Protocol | We transformed the plasmid pET-EC-SOD into the E. coli strain BL21(DE3). Cell Growth and Harvesting A Luria-Bertani (LB) medium (100ml containing 50µg/ml kanamycin) was inoculated with 50µl of glycerol stock of E. coli host cells and cultured overnight at 37 oC 250rpm. The overnight culture was inoculated into 1l of LB with 50µg/ml kanamycin and distributed aseptically into 2-l flasks (500ml per flask). The cells were cultured at 37 oC at 250rpm. The culture was then induced, after reaching an optical density of 0.6 at 600nm, by the addition of IPTG to a final concentration of 1mM and cultured at 37 oC, 250rpm for 3h. The culture was centrifuged at 6,000×g for 30min, the supernatant was carefully removed, and the cell pellet was washed by gently suspending in Tris buffer (100mM Tris, pH 8.0, 10mM EDTA, 100mM NaCl). The washed cell mass was collected by centrifuging at 14,900×g, 4 oC for 30min and stored at 20 oC. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding- Immobilized Metal affinity chromatography (IMAC) |
| Wash Buffer | Tris buffer (100mM Tris, pH 8.0, 10mM EDTA, 100mM NaCl |
| Solubilization Buffer | 50mM Tris-HCl (pH 8.0) containing 8M urea and 0.1M NaCl |
| Refolding Buffer | 50mM Tris-HCl, pH 8.0, 3mM GSH, 0.5mM GSSG, 50µM ZnCl2, 100µM CuSO4, and 1M urea |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 22.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 3/0.5 mM |
| Refolding Protocol | Immobilized Metal Affinity Chromatography The immobilized metal affinity chromatography (IMAC) was performed with HisTrap FF crude columns (GE Biosciences). The protein sample above was loaded onto the IMAC column, which was pre-equilibrated with equilibration buffer (50mM Tris- HCl, pH 8.0, 3mM GSH, 0.5mM GSSG, 8.0M urea) at a flow rate of 1ml/min, and then the column was washed again with 10ml of the equilibration buffer until the UV absorbance baseline was reached. Then, the refolding of the bound protein was performed on-column by the use of a linear urea gradient from 8.0M to 1M, starting with the equilibration buffer and finishing with a buffer containing 50mM Tris-HCl, pH 8.0, 3mM GSH, 0.5mM GSSG, 50µM ZnCl2, 100µM CuSO4, and 1M urea. The total gradient volume was 30ml and the flow rate was 0.6ml/min. The refolded rEC-SOD was eluted using renaturation buffer incorporating 1M urea and 1M imidazole, pH 8.0. We pooled the eluae and concentrated it by ultrafiltration using Vivaspin(MWCO=5,000). Gel Filtration Chromatography After the refolding step, the concentrated rEC-SOD was purified on a Superose 12 PC 3.2/30 column equilibrated with PBS buffer. Conalbumin (Mw=75,000), ovalbumin (Mw=43,000), carbonic anhydrase (Mw=29,000), and ribonuclease (Mw=13,700) were also loaded onto the column as calibration standards. The profile of rEC- SOD was then compared with the standards for molecular weight confirmation and the determination of multimer formation. |
| Refolding Assay | Western Blot,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 90% |
| Purity | n/a |
| Notes | n/a |