Refolding Record:
| Protein | |
|---|---|
| Protein Name | Interleukin-6 |
| Abbreviated Name | IL-6 |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P05231 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | aa.23-168 (Delta22) |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 146 |
| Molecular Weight | 16585.8 |
| Pi | 5.27312 |
| Molecular Weight | 16585.8 |
| Disulphides | 2 |
| Full Sequence |
SERIDKQI RYILDGISAL RKETSNKSNM SESSKEALAE NNLNLPKMAE KDGSFQSGFN EETSLVKIIT GLLEFEVYLE YLQNRFESSE EQARAVQMST KVLIQFLQKK AKNLDAITTP DPTTNASLLT KLQAQNQWLQ DMTTHLIL
|
| Notes | Other truncated versions of IL6 expressed and refolded: Delta 22Cys1,2 SERIDKQI RYILDGISAL RKETCNKSNM CESSKEALAE NNLNLPKMAE KDGSFQSGFN EETSLVKIIT GLLEFEVYLE YLQNRFESSE EQARAVQMST KVLIQFLQKK AKNLDAITTP DPTTNASLLT KLQAQNQWLQ DMTTHLIL Number of amino acids: 146 Molecular weight: 16617.9 Theoretical pI: 5.27 Delta 22Cys3,4 SERIDKQI RYILDGISAL RKETSNKSNM SESSKEALAE NNLNLPKMAE KDGCFQSGFN EETCLVKIIT GLLEFEVYLE YLQNRFESSE EQARAVQMST KVLIQFLQKK AKNLDAITTP DPTTNASLLT KLQAQNQWLQ DMTTHLIL Number of amino acids: 146 Molecular weight: 16617.9 Theoretical pI: 5.27 |
| Expression | |
|---|---|
| Report | Skelly SM, Tackney C, Hicklin D, Tamkins T, Goldstein N, Waksal H, Dagan S (1994) J Biotechnology, 34, 79-86 |
| Project Aim | Structure-Function |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pKK233-2 |
| Expression Protocol | 100ml cells cultures were grown in M9 media supplemented with casamino acids and thiamine. When A600 reached 0.4, expression was induced with 1mM IPTG for 4h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.4 |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 20mM TrisHCl pH 8.0, 50mM NaCl, 10mM EDTA, 0.5% Triton X-100 |
| Solubilization Buffer | 6M GdnHCl, 1mM EDTA, 0.1mM PMSF, 50mM TrisHCl pH 8.5 |
| Refolding Buffer | 20mM TrisHCl pH 8.5, 100mM NaCl, 1mM EDTA, 0.1mM PMSF |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cultures were harvested by centrifugation (3000rpm) and the pellet was resuspended in wash buffer containing 0.1mM PMSF and incubated with 300microg/ml lysozyme on ice for 30min. The lysate was homogenized on ice and then centrifuged (10000g, 30min). The pellet was washed twice in wash buffer, then resuspended in solubilization buffer and incubated at room temperature for 2h. The solution was then centrifuged (15000g, 1h). The supernatent was then refolded by extensive dialysis against refolding buffer. The insoluble material was removed by centrifugation and the soluble protein was then purified further using a Vydac C4 reverse-phase column. |
| Refolding Assay | ELISA |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 26% |
| Purity | |
| Notes | |