Refolding Record:
| Protein | |
|---|---|
| Protein Name | Growth Hormone |
| Abbreviated Name | GH |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01241 |
| 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 | 191 |
| Molecular Weight | 22129.0 |
| Pi | 5.268 |
| Molecular Weight | 22129.0 |
| Disulphides | 2 |
| Full Sequence |
FPTI PLSRLFDNAM LRAHRLHQLA FDTYQEFEEA YIPKEQKYSF LQNPQTSLCF SESIPTPSNR EETQQKSNLE LLRISLLLIQ SWLEPVQFLR SVFANSLVYG ASDSNVYDLL KDLEEGIQTL
MGRLEDGSPR TGQIFKQTYS KFDTNSHNDD ALLKNYGLLY CFRKDMDKVE TFLRIVQCRS VEGSCGF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Patra AK, Mukhopadhyay R, Mukhija R, Krishnan A, Garg LC, Panda AK. (2000) Protein Expression and Purification, 18, 182-192 |
| Project Aim | Undefined |
| Fusion | None |
| 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 | 4h |
| Expression Vector | pQE-60 |
| Expression Protocol | For large-scale production of r-hGH, recombinant E. coli cells were grown in a 3.5-L fermenter (2-L working volume) in complex medium. The initial glucose and yeast extract concentrations were 10 g/L. Fermentation was carried out at 37degC with vigorous aeration and agitation and the pH of the medium was maintained at 7 by use of 5 N NaOH. After 3 h of batch growth, the cells were grown in a fed-batch mode with a continuous supply of glucose and yeast extract. The culture at OD600 of 40 was induced with 1 mM IPTG, cultivated for another 4 h, and then harvested. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 40 |
| Cell Disruption Method | Sonication/French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | not stated |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris-HCl pH 8.0, 5 mM EDTA , 2% deoxycholate |
| Solubilization Buffer | 100mM Tris-HCl, 2M urea, pH12.5 |
| Refolding Buffer | 20mM Tris-HCl, 0.4M urea |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Pure inclusion bodies were isolated (104 mg protein isolated from 65 ml of high cell density fermentation broth) and dissolved in 16 ml of solubilization buffer. The solubilized r-hGH was diluted five times with Milli-Q water and the pH was brought down to 8.5 by adding 1 N HCl. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 50% |
| Purity | >90% |
| Notes | a range of solubilization buffers tested. |