Refolding Record:
| Protein | |
|---|---|
| Protein Name | Urodilatin |
| Abbreviated Name | rhudilatin |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 32 |
| Molecular Weight | 3525.1 |
| Pi | 11.3 |
| Molecular Weight | 3525.1 |
| Disulphides | Unknown |
| Full Sequence |
TAPRSLRRSSCFGGRMDRIGAQSGLGCMSFRY
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Sun Z, Xia Z, Bi F, Liu JN. (2008) Appl microbiol biotechnol, 1, 1 |
| Project Aim | Recombinant Protein Expression |
| Fusion | N-terminal small ubiquitin-related modifier (SUMO) |
| 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 | 3.5 h |
| Expression Vector | pET28 |
| Expression Protocol | Expression of SUMO-urodilatin fusion protein The E. coli strain BL21(DE3) was transformed by expression plasmid pET28/SUMO-urodilatin. A single-colony transformant was inoculated into Luria–Bertani (LB) medium containing 25 µg/ml kanamycin and grown overnight at 37°C. The culture was transferred to fresh LB medium supplemented with kanamycin and allowed to grow at 37°C until the optical density (OD600) reached about 0.8. Isopropylthiogalactoside (IPTG) was then added to a final concentration of 0.5 mM to induce expression of SUMO-urodilatin fusion protein, and the cultivation was continued at 37°C for 3.5 h or at 25°C for 6 h. The cells were harvested by centrifugation at 5,000×g for 10 min. Total proteins in bacteria lysate were applied to 13% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, and the expression yield was analyzed using the Quantity One Quantitation software (Bio-Rad) according to the relative band intensities of Coomassie blue stain. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.8 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 20 mM Tris–HCl, 8 M urea, 0.5 M NaCl, 20 mM imidazole, 10 mM 2-mercaptomethanol, pH 8.0 |
| Solubilization Buffer | 20 mM Tris–HCl, 8 M urea, 0.5 M NaCl, 200 mM imidazole, 10 mM 2-mercaptomethanol, pH 8.0 |
| Refolding Buffer | 50 mM Tris–HCl, 2.5 M urea, 1.0 mM ethylenediaminetetraacetic acid, pH 8.5 |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.5 |
| Refolding Temperature | 10.0 |
| Protein Concentration | n/a |
| Refolding Time | 20 h |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 10 mM |
| Refolding Protocol | About 8.4 g Bacteria pellets harvested from 2 l culture medium were suspended in 60 ml binding buffer (20 mM Tris–HCl, 6 M guanidine HCl, 0.5 M NaCl, 5 mM imidazole, 10 mM 2-mercaptomethanol, pH 8.0) and disrupted by pulse sonication in ice/water bath. The lysate was centrifuged at 12,000×g for 25 min at 4°C, and the supernatant was applied to a Ni-sepharose column, which was packed with 10 ml resin and pre-equilibrated with binding buffer. The column was washed sequentially with 150 ml binding buffer and then 100 ml wash buffer (20 mM Tris–HCl, 8 M urea, 0.5 M NaCl, 20 mM imidazole, 10 mM 2-mercaptomethanol, pH 8.0). The fusion protein was eluted with 50 ml elution buffer (20 mM Tris–HCl, 8 M urea, 0.5 M NaCl, 200 mM imidazole, 10 mM 2-mercaptomethanol, pH 8.0). Renaturation was performed by diluting 25 ml purified SUMO-urodilatin fusion protein (∼3.5 mg/ml) with 500 ml refolding buffer (50 mM Tris–HCl, 2.5 M urea, 1.0 mM ethylenediaminetetraacetic acid, pH 8.5). After incubation at 10°C for 20 h, the refolding sample was extensively dialyzed against the digestion buffer (50 mM Tris–HCl, 0.1 M NaCl, pH 7.5). The precipitation was removed by centrifugation at 12,000×g for 20 min at 4°C, and the supernatant was concentrated to about 1.5 mg/ml by ultrafiltration with a 10 kDa cut-off membrane. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |