Refolding Record:
| Protein | |
|---|---|
| Protein Name | Interleukin-21 |
| Abbreviated Name | IL-21 |
| SCOP Family | Short Chain Cytokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q9HBE4 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 132 |
| Molecular Weight | 15393.6 |
| Pi | 9.41442 |
| Molecular Weight | 15393.6 |
| Disulphides | Unknown |
| Full Sequence |
MAQDRHMI RMRQLIDIVD QLKNYVNDLV PEFLPAPEDV ETNCEWSAFS CFQKAQLKSA NTGNNERIIN VSIKKLKRKP PSTNAGRRQK HRLTCPSCDS YEKKPPKEFL ERFKSLLQKM IHQHLSSRTH GSED
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Asano R, Kudo T, Makabe K, Tsumoto K, Kumagai I (2002) FEBS Letters, 528, 70-76 |
| Project Aim | Functional Studies |
| 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 | overnight |
| Expression Vector | pUT |
| Expression Protocol | Cells were incubated in LB medium at 37degC. When OD600 reached 0.8, expression was induced by the addition of 1mM IPTG and the cells were grown overnight. Cells were harvested by centrifugation (2000g x 35min) and then resuspended in 10ml phosphate-buffered saline (PBS). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.8 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M GdnHCl, phosphate buffered saline (PBS) |
| Refolding Buffer | 50mM TrisHCl, 200mM NaCl, 1mM EDTA pH 8.0 |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 36-72h |
| Redox Agent | GSH |
| Redox Agent Concentration | 3.75mM |
| Refolding Protocol | Cells were sonicated (150W for 15min) and centrifuged (20min x 4500g). The insoluble fraction was dissolved in solubilization buffer overnight at 4degC. Following solubilization, the proteins were purified using a Talon metal affinity resin column. Following purification, the protein was refolded by a step-wise dilution procedure. Purified protein was diluted to 7.5micromolar with 6M GdnHCl, 50mM TrisHCl (pH 8.0) 375micromolar 2-mercaptoethanol, then dialysed step-wise through a series of refolding buffers containing decreasing amounts of GdnHCl (3M,2M,1M,0.5M,0M GdnHCl). At low concentrations of GdnHCl (0.5M, 1M), 0.4M L-arginine was included in the buffer, as was 3.75mM GSH and 375micromolar GSSG for disulfide reshuffling. Each dialysis step lasted 6-12 hours. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | >200mg/L |
| Purity | |
| Notes | - Refolding yield increased from 57% with oxidizing reagent alone to 90% with oxidizing and reducing reagents |