Refolding Record:
| Protein | |
|---|---|
| Protein Name | ZEBRA protein also known as Z protein |
| Abbreviated Name | ZEBRA |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Epstein-Barr virus |
| UniProt Accession | Q3KSS8 |
| 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 | 26813.2 |
| Pi | 5.24 |
| Molecular Weight | 26813.2 |
| Disulphides | Unknown |
| Full Sequence |
MMDPNSTSEDVKFTPDPYQVPFVQAFDQATRVYQDLGGPSQAPLPCVLWPVLPEPLPQGQLTAYHVSAAPTGSWFPAPQPAPENAYQAYAAPQLFPVSDITQNQLTNQAGGEAPQPGDNSTVQPAAAVVLACPGANQEQQLADIGAPQPAPAAAPARRTRKPLQPESLEECDSELEIKRYKNRVASRKCRAKFKHLLQHYREVASAKSSENDRLRLLLKQMCPSLDVDSIIPRTPDVLHEDLLNF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | El-Guindy AS, Paek SY, Countryman J, Miller G. (2006) Biologycal Chemistry, 281, 3085-3095 |
| Project Aim | Expression and identification |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 2 h |
| Expression Vector | pET22b |
| Expression Protocol | Escherichia coli (BL21) cells were transformed with pET22b/cDNA-Z or pET22b/cDNA-Z(T14A). ZEBRA expression was induced by growing the cells in Luria-Bertani (LB) medium containing 1 mM isopropyl -D-thiogalactopyranoside for 2 h at 37°C. The induced culture was centrifuged at 5000 x g for 10 min at 4 °C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 5 mM imidazole, 0.5 M NaCl, and 20 mM Tris-HCl (pH 7.9) |
| Solubilization Buffer | 6 M urea |
| Refolding Buffer | decreasing concentrations of urea |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 0.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The pellet was re-suspended in nickel column binding buffer containing 5 mM imidazole, 0.5 M NaCl, and 20 mM Tris-HCl (pH 7.9). ZEBRA was extracted by sonicating the cell lysate in binding buffer containing 6 M urea. The cell extract was cleared by centrifugation at 20,000 x g for 15 min at 4 °C, and the supernatant was loaded on a nickel affinity column. The retained ZEBRA protein was eluted by a stepwise gradient of imidazole ranging from 0 to 1 M dissolved in 10 mM Tris-HCl (pH 7.9), 0.25 M NaCl, and 6 M urea. Column fractions containing the ZEBRA protein were pooled; the purity of ZEBRA was examined by silver nitrate staining of an SDS-polyacrylamide gel. Purified ZEBRA proteins were refolded by dialysis against gradually decreasing concentrations of urea. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |