Refolding Record:
| Protein | |
|---|---|
| Protein Name | Casein kinase II subunit alpha-1-subunit beta-1 |
| Abbreviated Name | CKA1-CKB1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Arabidopsis thaliana |
| UniProt Accession | Q08467-P40228 |
| 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 | 621 |
| Molecular Weight | 71595.1 |
| Pi | 6.27 |
| Molecular Weight | 71595.1 |
| Disulphides | Unknown |
| Full Sequence |
MSKARVYTEVNVIRPKDYWDYESLIVQWGEQDDYEVVRKVGRGKYSEVFEGINVNSKEKCIIKILKPVKKKKIRREIKILQNLCGGPNIVKLLDVVRDQHSKTPSLIFEYVNSTDFKVLYPTLTDYDIRYYIYELLKALDFCHSQGIMHRDVKPHNVMIDHELRKLRLIDWGLAEFYHPGKEYNVRVASRYFKGPELLVDLQDYDYSLDMWSLGCMFAGMIFRKEPFFYGHDNQDQLVKIAKVLGTDELNAYLNKYQLELDPQLEALVGRHSRKPWSKFINADNQHLVSPEAIDFLDKLLRYDHQDRLTAKEAMAHAYFAQVRAAETSRMRSQMYRDRGTVNSRPEVVDRKRINDALERPSPSTSRQVNGKGKGTVTAATTTANLIGKQQSNNINHRDSRSASLSKNNTVSDDESDTDSEESDVSGSDGEDTSWISWFCNLRGNEFFCEVDDDYIQDDFNLCGLSSLVPYYEYALDLILDVESSQGEMFTEEQNELIESAAEMLYGLIHARYILTSKGLAAMLDKYKNYDFGRCPRVYCCGQPCLPVGQSDLPRSSTVKIYCPKCEDIYYPRSKYQGNIDGAYFGTTFPHLFLMTYGHLKPAKATQNYVQRVFGFKLHKP
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Klimczak LJ, Collinge MA, Farini D, Giuliano G, Walker JC, Cashmore AR. (1995) Plant Cell., 7, 105-115 |
| 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)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pET11d |
| Expression Protocol | Expression strains were grown in Luria-Bertani medium at 37°C to an OD of 0.6 to 0.8, IPTG was added to the culture to the final concentration of 0.4 mM, and incubation was continued for 3 hr. Cells were collected by centrifugation, washed in 0.9% (w/v) NaCI, and lysed in a 1150th culture volume of TE buffer (10 mM Tris-HCI, pH 8.0, 1 mM EDTA) (the CKB1-expressing strain was incubated for 10 min on ice in the presence of 20 pg1mL lysozyme; for the CKAl strain, the addition of exogenous lysozyme was not necessary). After lysis by sonication, the lysates were centrituged for 30 min at lOO,OOOg, and the supernatant was assayed for protein kinase activity. This lysate is referred to as the low-salt extract. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6-0.8 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M urea. 0.1 M DTT |
| Refolding Buffer | 100 mM Tris-HCI. pH 8.0. 0.4 M arginine. 2 mM EDTA. 4.5 mM DTT. and 1 mM oxidized glutathione |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | GSSG/DTT |
| Redox Agent Concentration | 1/4.5 mM |
| Refolding Protocol | The protocols for solubilization and renaluration of proteins were based on those published by Lin and Traugh (1993). lnclusion bodies were solubilized for 2 hr at room temperature in 1166th culture volume of 8 M urea. 0.1 M DTT. Renaturation was performed by diluting the solubilized protein 66-fold in a renaturation buffer composed of 100 mM Tris-HCI. pH 8.0. 0.4 M arginine. 2 mM EDTA. 4.5 mM DTT. and 1 mM oxidized glutathione and by incubating overnight at 4°C. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.4 M |
| Refolding Yield | n/a |
| Purity | 78 mg |
| Notes | n/a |