Refolding Record:
| Protein | |
|---|---|
| Protein Name | Fibrinolytic enzyme |
| Abbreviated Name | F-III-2 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Lumbricus rubellus (Humus earthworm) |
| UniProt Accession | Q9BLI8 |
| 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 | 245 |
| Molecular Weight | 26155.2 |
| Pi | 4.61 |
| Molecular Weight | 26155.2 |
| Disulphides | Unknown |
| Full Sequence |
MELPPGKIVGGIEARPYEFPWQVSVRRKSSDSHFCGGSIINDRWVVCAAHCMQGESPALVSLVVGEHDSSAASTVRQTHDVDSIFVNENYDPRTLENDVSVIKTAIAITFDINVGPICAPDPANDYVYRKSQCSGWGTINSGGICCPAVLRYVTLNITTNAFCDAVYTSDTIYDDMICATDNTGMTDRDSCQGDSGGPLSVKDGSGIFSLGGIVSWGIGCASGYPGVYSRVGFHAGWITDTITNN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Li DH, Tong W, Yang YF (2008) WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY, 24, 613-618 |
| Project Aim | Over expression & Renaturation |
| 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(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 5 h |
| Expression Vector | pET28a |
| Expression Protocol | E. coli BL21 (DE3) pLysE cells containing a plasmid of interest were grown at 37 °C for 12 h in 10 ml Luria-Bertani (LB) medium containing 50 μg/ml of kanamycin. The culture was diluted 1:100 (v/v) into 400 ml of the same medium and grown at 37 °C until an OD600 of 0.5 was reached. Then a final concentration (1.0 mM) of isopropyl β-D-thiogalactopyranoside (IPTG) was added into the culture, and induction was at 37 °C for 5 h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 50 mM Tris-HCl buffer (pH 7.5), containing 8 M urea, 1 mM PMSF and 10 mM dithiothreitol (DTT) |
| Refolding Buffer | 50 mM Tris-HCl buffer, 3 mM reduced glutathione, 1 mM oxidized glutathione, pH 7.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1/3 mM |
| Refolding Protocol | Induced bacteria were collected by centrifugation and suspended in 50 mM Tris-HCl buffer (pH 7.5). Then, the bacteria cell lysate was prepared by sonication at 4 °C. The supernatant was removed by centrifugation at 12,000 g for 10 min, and the pellet (inclusion bodies) was used for purification. The inclusion bodies were dissolved in 50 mM Tris-HCl buffer (pH 7.5), containing 8 M urea, 1 mM PMSF and 10 mM dithiothreitol (DTT). Centrifugation at 12,000 g for 10 min took place to collect the supernatant for 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To gradually decrease the concentration of urea in the protein solution, the protein refolding was processed by dialysis at 4 °C against the buffer systems (50 mM Tris-HCl buffer, 3 mM reduced glutathione, 1 mM oxidized glutathione, pH 7.5), containing 6, 4, 3, 2, 1 and 0.5 M urea, respectively, and finally, the residual urea was eliminated by dialysis against the same buffer, but containing no urea. |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |