Refolding Record:
| Protein | |
|---|---|
| Protein Name | Daniplestim (interleukin-3 variant) |
| Abbreviated Name | SC-55494 |
| SCOP Family | Short Chain Cytokines |
| Structure Notes | |
| Organism | Unknown |
| UniProt Accession | P08700 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 111 |
| Molecular Weight | 12591.5 |
| Pi | 5.46357 |
| Molecular Weight | 12591.5 |
| Disulphides | 1 |
| Full Sequence |
ANCSIMIDEIIHHLKRPPNPLLPNNLNSEDMDILMERNLDTPNLLA
FVRAVKHLENASGIEAILRNLQPCLPSATAAPSRHPIIIKAGDWQE
FREKLTFYLVTLDQAQEQQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Boyle DM, McKinnie RE, Joy WD, Violand BN, McLaughlin JK, Landis BH. (1999) Biotechnol. Appl. Biochem., 30, 163-170 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | K12 W3110 |
| Expression Temp | 37.0 |
| Expression Time | 4 |
| Expression Vector | recA promotor |
| Expression Protocol | Cells were grown in M9+2% casamino acids medium containing 50mg/ml spectinomycin with glucose concentrations maintained between 2 and 6g/L. The pH of the culture was maintained at 7.0 by addition of NH4OH. When OD550 reached 13.5, expression was induced by the addition of nalidixic acid, and the culture was grown for a further 4h. After incubation the culture was cooled to 15degC and centrifuged. The cells were then stirred overnight in 10mM Tris/EDTA pH 8.0 at 10degC. |
| Method of Induction | Nalidixic Acid |
| Cell Density at Induction | OD 550 = 13.5 |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | not stated |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 30mM Tris pH 9.0, +/-2mM cysteine, 8M Urea or 6M GdnHCl |
| Refolding Buffer | 2M GdnHCl, 0.5mM cysteine, pH 9.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 9.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 48h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | n/a,0.5mM |
| Refolding Protocol | The inclusion bodies were dissolved in solubilization buffer and mixed for 30min at 4degC. The protein was refolded by dialysis at 4degC for 48h |
| Refolding Assay | HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | various refolding conditions tested, including the use of urea and DTT (see paper for more details) |