Refolding Record:
| Protein | |
|---|---|
| Protein Name | S-phase delaying protein 1 |
| Abbreviated Name | Spd1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Schizosaccharomyces pombe (Fission yeast) |
| UniProt Accession | Q10585 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 124 |
| Molecular Weight | 14225.0 |
| Pi | 6.73 |
| Molecular Weight | 14225.0 |
| Disulphides | Unknown |
| Full Sequence |
MHSSKRVMTTKTHVEQPESSMRPQLPESIQGSLMDVGMRVRKSISTGYKSKQTTFPAYNPPLYNTVSENIALKNTAFSYEPNGTKRPFEQAIPNYNWANPPQDFEEPEWLKPFDVVMEGTNERL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Håkansson P, Dahl L, Chilkova O, Domkin V, Thelander L. (2006) Biologycal Chemistry, 281, 1778-1783 |
| 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 | pET3a |
| Expression Protocol | The Spd1p-expressing bacteria were grown the same way as the Suc22p-expressing bacteria but at 37 °C, and they were induced with isopropyl 1-thio--D-galactopyranoside at a final concentration of 0.5 mM. After lysing the bacteria by freezing and thawing, the crude extract was centrifuged for 60 min at 45,000 rpm at 4 °C in a Beckman Ti-70 rotor, and the supernatant was discarded |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Freeze-thaw |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris-Cl, pH 7.4, treated with DNase 1 (5 µg/ml) |
| Solubilization Buffer | 0.1 M Tris-Cl, pH 8.5, 5 M urea |
| Refolding Buffer | 50 mM Tris-Cl, pH 7.5, 100 mM potassium acetate |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The pellet was washed three times with 50 mM Tris-Cl, pH 7.4, treated with DNase 1 (5 µg/ml), and dissolved in 0.1 M Tris-Cl, pH 8.5, 5 M urea. After incubation for 20 min on ice, the solution was centrifuged at 45,000 x g for 20 min at 4 °C and the pellet discarded. Finally, the urea-solubilized Spd1p was refolded by instant 10x dilution in 50 mM Tris-Cl, pH 7.5, 100 mM potassium acetate; the remaining urea was removed by gel filtration on a Sephadex G50 column equilibrated with the same buffer. |
| Refolding Assay | RNR assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |