Refolding Record:
| Protein | |
|---|---|
| Protein Name | Growth Hormone |
| Abbreviated Name | GH |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Sparus aurata (Gilthead seabream) |
| UniProt Accession | Q90YK4 |
| 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 | 187 |
| Molecular Weight | 21230.2 |
| Pi | 6.96 |
| Molecular Weight | 21230.2 |
| Disulphides | 0 |
| Full Sequence |
MAQPITDGQRLFSIAVSRVQHLHLLAQRLFSDFESSLQTEEQPQLNKIFLQDFCNCDYIISPIDKHETQRSSVLKLLSISYRLVESWEFPSRSLSGGSAPRNQISPKLSELKTGIHLLIRANEDGAEIFPDRSALQLAPYGNYYQSLGTDESLRRTYELLACFKKDMHKVETYLTVAKCRLSSVGST
|
| Notes | Sequence refers to GH fragment altered using specific primers prior to insertion into pET8 plasmid as no details on plasmid could be found. |
| Expression | |
|---|---|
| Report | Ben-Atia, I., Fine, M., Tandler, A., Funkenstein, B., Maurice, S., Cavari, B., Gertler, A., (1999) Gen Comp Endocrinol., 113, 155-164 |
| Project Aim | Functional Studies,Drug Studies,Recombinant Protein Expression |
| 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 | 4h |
| Expression Vector | pET-8 |
| Expression Protocol | Cells grown in 400ml TB medium in 2.5 L flasks prior to induction. After incubation cells were harvested by centrifugation(10,000, 10min) and stored at -20degC. Frozen cells were then thawed and resuspended in 200ml cold resuspension buffer(10mM EDTA, 10mM TrisHCl, pH 8.0) in the presence of lysozyme(0.5mg/ml) and incubated(30min, 4degC). Cells were then sonicated prior to centrifugation(25,000g, 30min.) Inclusion bodies were further sonicated in distilled water and resuspended several times untill a clean pellet was obtained. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.9 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | n/a |
| Solubilization Buffer | 4.5 M urea, 40mM Tris base, pH 11.3, 0.1 mM cysteine. |
| Refolding Buffer | 10mM TrisHCl, pH 8.0 |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 0.1mM |
| Refolding Protocol | Inclusion bodies solubilized in 300ml solubilization buffer and solution stirred at 4degC for 1h. The solution was then diluted with 2 vol. of cold water and dialyzed for 48h against 4x10L refolding buffer. This was then applied to a Q Sepharose column, preincubated with the refolding buffer at 4degC. Elution was carried out using a discontinuous NaCl gradient in refolding buffer. |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 280nm |
| Purity | n/a |
| Notes | n/a |