Refolding Record:
| Protein | |
|---|---|
| Protein Name | Somatotropin also known as Growth hormone |
| Abbreviated Name | GH |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Ovis aries (sheep/ovine) |
| UniProt Accession | P67930 |
| 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 | 190 |
| Molecular Weight | 21787.0 |
| Pi | 7.82 |
| Molecular Weight | 21787.0 |
| Disulphides | 2 |
| Full Sequence |
FPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDVTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTETYLRVMKCRRFGEASCAF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Khan RH, Rao KB, Eshwari AN, Totey SM, Panda AK. (1998) Biotechnol Prog, 1998, 722-728 |
| Project Aim | Over expression & Renaturation |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | M15 |
| Expression Temp | 37.0 |
| Expression Time | 5-6 h |
| Expression Vector | pQE-30 |
| Expression Protocol | The fermentation was carried out at 37 °C with vigorous aeration and agitation and at OD600 ) 10 were induced with 1 mM IPTG and cultivated for another 5-6 h. The harvested cells were checked for expression and processed for the purification of inclusion bodies. Samples were collected at an interval of 30 min during the fermentation to check cell growth, glucose consumption, and protein expression. For the analysis of protein concentration during different stages of growth, inclusion bodies were separated from the cell and purified and then dissolved in 1% SDS solution in 50 mM Tris-HCl buffer at pH 8. Protein content was determined by BCA assay and growth hormone levels by RIA. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 10 = 600 |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris-HCl buffer (pH 8.0) containing 5 mM EDTA and 2% deoxycholate solution |
| Solubilization Buffer | 2 M Tris buffer pH 12 and urea |
| Refolding Buffer | 50 mM Tris-HCl at pH 8 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 100 µg |
| Refolding Time | 10-12 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Refolding of Recombinant oGH. Recombinant oGH after solubilization at pH 12 was refolded by two methods. In the first method, the solubilized protein was diluted 50-fold in 50 mM Tris-HCl at pH 8 and stirred at room temperature for 10-12 h. |
| Refolding Assay | Western Blot,SDS-PAGE,Receptor binding,Circular Dichroism (uv-CD) |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 40% |
| Purity | n/a |
| Notes | n/a |