Refolding Record:
| Protein | |
|---|---|
| Protein Name | NS2B-NS3 Protease |
| Abbreviated Name | DEN2 NS2B-NS3 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Japanese encephalitis virus |
| UniProt Accession | P32886 |
| 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 | 317 |
| Molecular Weight | 34365.2 |
| Pi | 5.0 |
| Molecular Weight | 34365.2 |
| Disulphides | Unknown |
| Full Sequence |
GWPATEFLSAVGLMFAIVGGLAELDIESMSIPFMLAGLMAVSYVVSGKATDMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVPWKVWVLRMSCIGLAALTPWAIVPAAFGYWLTLKTTKRGGVFWDTPSPKPCSKGDTTTGVYRIMARGILGTYQAGVGVMYENVFHTLWHTTRGAAIMSGEGKLTPYWGSVKEDRIAYGGPWRFDRKWNGTDDVQVIVVEPGKAAVNIQTKPGVFRTPFGEVGAVSLDYPRGTSGSPILDSNGDIIGLYGNGVELGDGSYVSAIVQGDRQEEPVPEAYTPNMLRK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lin CW, Lin KH, Lyu PC, Chen WJ. (2006) Virus Res., 116, 106-113 |
| Project Aim | Undefined |
| 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 | 20.0 |
| Expression Time | 4 h |
| Expression Vector | pET24a |
| Expression Protocol | The transformed E. coli BL21(DE3) cells with pET24a-NS2B-NS3 were grown in LB medium containing 100 μg of ampicillin and grown at 37 °C. Once the cells reached an absorbance at 600 nm of 0.6, they were induced by the addition of 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 20 °C for 4 h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 10 mM imidazole, 8 M urea and 1 mM β-mercaptoethanol |
| Refolding Buffer | 10 mM imidazole and 1 mM β-mercaptoethanol |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 0.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 1 mM |
| Refolding Protocol | Finally, the bacteria were harvested by centrifugation at 9000 rpm for 15 min at 4 °C, and resuspended in the denature buffer (10 mM imidazole, 8 M urea and 1 mM β-mercaptoethanol) for sonication. The supernatant after centrifugation (10,000 × g for 20 min) was applied into the His-Bind Resin column (Novagen). For decreasing concentrations of urea, recombinant NS2B-NS3 protease slowly renatures in-gel with a gradient washing by the refolding buffer (10 mM imidazole and 1 mM β-mercaptoethanol) at 4 °C overnight. After washing with 100 mM imidazole, recombinant NS2B-NS3 protease was eluted using 400 mM imidazole, and then further dialyzed against phosphate-buffered saline. |
| Refolding Assay | Western Blot,SDS-PAGE,ELISA |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |