Refolding Record:
| Protein | |
|---|---|
| Protein Name | Growth Hormone |
| Abbreviated Name | GH |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Pig (Sus scrofa) |
| UniProt Accession | P01248 |
| 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 | 192 |
| Molecular Weight | 21933.1 |
| Pi | 6.83097 |
| Molecular Weight | 21933.1 |
| Disulphides | 2 |
| Full Sequence |
MAFPAM PLSSLFANAV LRAQHLHQLA ADTYKEFERA YIPEGQRYSI QNAQAAFCFS ETIPAPTGKD EAQQRSDVEL LRFSLLLIQS WLGPVQFLSR VFTNSLVFGT SDRVYEKLKD LEEGIQALMR ELEDGSPRAG QILKQTYDKF DTNLRSDDAL LKNYGLLSCF KKDLHKAETY LRVMKCRRFV ESSCAF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Puri NK (1991) FEBS Letters, 292, 187-190 |
| Project Aim | Folding |
| 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 | not stated |
| Expression Vector | pMG93 |
| Expression Protocol | no detail provided |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 0.1M citric acid, 0.2M disodium phosphate pH 4.0 |
| Solubilization Buffer | 0.1M TrisHCl pH 10.0, 2% (v/v) mercaptoethanol, 5% (w/v) cetyltimethylammonium chloride |
| Refolding Buffer | 20mM ethanolamineHCl pH 10.0, various concentrations of 2-mercaptoethanol |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 10.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 1.5-7.5mg/ml |
| Refolding Time | 24-48h |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | various concentrations |
| Refolding Protocol | Inclusion bodies were isolated by cell disruption and harvested by differential centrifugation. Inclusion bodies were then washed twice with wash buffer and twice with distilled water. Approximately 50mg (117mg/ml dry weight) of inclusion bodies was solubilised at 10mg/ml in solubilization buffer for 1h at 55degC. The solubilised inclusion bodies were centrifuged (5min, 10000g) and the supernatant was mixed with 8 bed volumes of Dowex 50W x 4 ion exchange resin equilibrated in 0.1M glyucineHCl, 5M urea pH 10.0. The protein was passed down a G-25 sephadex gel filtration column into a solution of 20mM ethanolamineHCl pH 10.0 with final concentrations of 2-mercaptoethanol at 5,45,55,75 and 100mM. Refolding was performed for 24-48h with shaking at 4degC. |
| Refolding Assay | HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 12-28% |
| Purity | |
| Notes | Refolding by dialysis without 2-mercaptoethanol resulted in only 15% successful refolding. Refolding in different concentrations of 2-mercaptoethanol resulted in the following yields: 5mM 2-BME: 12% 45mM 2-BME: 25% 75mM 2-BME: 28% 100mM 2-BME: 24% 55mM 2-BME: 28% Optimal protein refolding at 55mM BME and 3.5mg/ml protein |