Refolding Record:
| Protein | |
|---|---|
| Protein Name | Casein kinase II subunit alpha |
| Abbreviated Name | CK II alpha subunit |
| SCOP Family | Protein kinases, catalytic subunit |
| Structure Notes | |
| Organism | Drosophila melanogaster (Fruit fly) |
| UniProt Accession | P08181 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 336 |
| Molecular Weight | 39828.5 |
| Pi | 6.71 |
| Molecular Weight | 39828.5 |
| Disulphides | Unknown |
| Full Sequence |
TLPSAARVYTDVNAHKPDEYWDYENYVVDWGNQDDYQLVRKLGRGKYSEVFEAINITTTEKCVVKILKPVKKKKIKREIKILENLRGGTNIITLLAVVKDPVSRTPALIFEHVNNTDFKQLYQTLTDYEIRYYLFELLKALDYCHSMGIMHRDVKPHNVMIDHENRKLRLIDWGLAEFYHPGQEYNVRVASRYFKGPELLVDYQMYDYSLDMWSLGCMLASMIFRKEPFFHGHDNYDQLVRIAKVLGTEELYAYLDKYNIDLDPRFHDILQRHSRKRWERFVHSDNQHLVSPEALDFLD
KLLRYDHVDRLTAREAMAHPYFLPIVNGQMNPNNQQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lin WJ, Traugh JA. (1993) Protein Expression and Purification, 4, 256-264 |
| Project Aim | Identification and Characterization |
| 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 | 37.0 |
| Expression Time | 2 h |
| Expression Vector | pET3a |
| Expression Protocol | The recombinant pT3a vectors were used to individually transform E. coli BL21(DE3). Routinely, the transformed bacteria were grown in 25 ml of M9ZB medium at 37 c. When the OD 600 reached 0.5-0.6, 0.4 mM IPTG was added to induced the expression of the alpha and beta subunits. After induced at 37 c for 2 h, the bacteria were harvested ,suspended in 1 ml of extract buffer ( 50 ml Tris-HCl, pH 7.4, 10 mM EGTA,10 mM 2 mercaptoethanol, 1 mM PMSF and 0.02% NaN3), and sonicated for three 15-s cycles on ice with a 1-min pause between pulses. The pellet which contain inclusion bodies was washed with, recentrifuged, and resuspended in 0.7 ml of extract buffer. The expressed alpha and beta subunits of casein kinase II were visualized with coomassie brilliant blue following polyacrylamide gel electrophoresis in sodium dodecyl sulfate. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5-0.6 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris–HCl, pH 7.5, 10 mM EGTA, 10 mM 2-mercaptoethanol, 1 mM PMSF,0.02% NaN3 |
| Solubilization Buffer | 8 M urea , 0.1 M dithiothreitol |
| Refolding Buffer | 100 mM Tris-HCl, pH 8.5, 2 mM EDTA, 0.02% NaN3, 0.2 mM PMSF |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | To recover soluble active recombinant alpha subunits from inclusion bodies, the aggregated alpha subunits were solubilized with 8 M urea and 0.1 M dithiothreitol in a final volume of 0.039 ml at room temperature for 2 h and renatured by diluting with renaturating buffer to a final volume of 2.6 ml. |
| Refolding Assay | Gel filtration chromatography |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |