Refolding Record:
| Protein | |
|---|---|
| Protein Name | Insulin |
| Abbreviated Name | Insulin |
| SCOP Family | Insulin-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01308 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | 31-residue linker C-peptide replaced with pentapeptide YPGDV |
| Chain Length | 60 |
| Molecular Weight | 6924.0 |
| Pi | 7.78 |
| Molecular Weight | 6924.0 |
| Disulphides | 2 |
| Full Sequence |
FVNQHL CGSHLVEALY LVCGERGFFY TPKTRR YPGDV KRG IVEQCCTSIC SLYQLENYCN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Chang S, Kim D, Choi K, Shin J, Shin H (1998) Biochem J., 329, 631-635 |
| Project Aim | Structural Studies |
| Fusion | N-terminal poly-histidine tag |
| 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 | not stated |
| Expression Vector | pET3a |
| Expression Protocol | not stated |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 1:50mM TrisHCl pH 9.0, 6M GdnHCl, 2:8M urea, 0.3M HCl, 4:7M urea, 20mM formic acid pH 4.0 |
| Refolding Buffer | 50mM glycine, pH 11.5 |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | yes |
| Refolding pH | 11.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 50microM |
| Refolding Time | 18h |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 100microM |
| Refolding Protocol | Cultured E.coli cells were harvested by centrifugation and resuspended in 20mM TrisHCl pH 8.0, 1mM EDTA, 1mM PMSF. After cell disruption by sonication, the lysate was centrifuged (10min, 6000g). The pellet was dissolved in solubilization buffer 1 at a protein concentration of 10mg/ml. Oxidative sulphitolysis was performed by the addition of 300mM sodium sulphite and 60mM sodium tetrathionate and incubation for 12h at room temperature. The proteins were precipitated by adding 2 volumes of 0.1M ZnCl2 and precipitates were collected by centrifugation (15min, 6000g). The precipitates were then dissolved in solubilization buffer 2 at 10mg/ml and cleaved by CnBr at room temperature for 16h in the dark. The proteins were precipitated again with 2 volumes of 0.5M ZnCl. The precipitates were then solubilized in solubilization buffer 3 and loaded onto an S-Sepharose cation-exchange buffer. The protein was eluted at 3ml/min using a linear gradient of 0-0.5M NaCl in solubilization buffer 3 for 50min. Selected fractions were pooled and 2 volumes of distilled water was added, after 30min at room temperature precipiates were collected by centrifugation (6000g, 15min). After washing with the same volume of water again, the protein was lyophilized and stored at -20degC. The protein was dissolved in 50mM glycine buffer at 50microM and 2 molar equivalents of 2-mercaptoethanol was added. After 18h at 4degC with gentle agitation, the reaction was stopped by acidification with H3PO4 to pH 2.5. |
| Refolding Assay | HPLC,Receptor binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 78% |
| Purity | n/a |
| Notes | n/a |