Refolding Record:
| Protein | |
|---|---|
| Protein Name | Insulin-like growth factor II |
| Abbreviated Name | IGF2 |
| SCOP Family | Insulin-like |
| Structure Notes | |
| Organism | Oreochromis mossambicus (Mozambique tilapia) |
| UniProt Accession | O73722 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 79 |
| Molecular Weight | 9084.3 |
| Pi | 6.56939 |
| Molecular Weight | 9084.3 |
| Disulphides | 0 |
| Full Sequence |
MA MEMA SAETLFGGKL VDALQFVCED RSFYFSRPTS RGNNRRPQTR GIVEECLFCS CDLNLLEQYC AKPAKSE HHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hu SY, Wu JL, Huang JH. (2004) J Biotechnology, 107, 161-171 |
| Project Aim | Structural Studies |
| Fusion | C-terminal hexahistidine 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 | 12h |
| Expression Vector | pET28b |
| Expression Protocol | Fed-batch cultivation was carried out at 37degC in a 5L bioreactor. The initial work volume was 1.30 l of modified R-medium, containing citric acid (3g/L), KH2PO4 (6.75g/L), (NH4)2SO4 (5g/L), Na2HPO4.12H2O (3g/L), MgCl2 (1.5g/L), NH4Cl (0.1g/L), glucose (20g/L), yeast extract (20g/L), N-Z-Amine AS (30g/L) and 6ml/L of a trace metal solution. The composition of trace metal solution that contains (per ml of 5M HCl) FeSO4.7H2O 10mg, ZnSO4.7H2O 2.25mg, CaCl2.2H2O 1.35mg, MnSO4.5H2O 0.5mg, CuSO4.5H2O 1mg, AlCl.6H2O 0.3mg, (NH4)6Mo7O24.4H2O 0.1mg, H3BO3 0.2mg, and thiamine–HCl 2mg. Glucose was sterilized separately and trace metal solution was sterilized by filtration. The entire content of seed culture (7.69% v/v) was inoculated. The agitation speed and flow rate of aeration was set at 1000 rpm and 3L/min. The pH was kept at 7.0 by adding 28% (v/v) ammonium water. The dissolved oxygen concentration was maintained above 20% of air saturation by increasing the percentage of pure oxygen using a gas mixer. A nutrient feeding solution was added by using the pH-stat feeding strategy. The nutrient solution contained glucose 750g/L, yeast extract 50g/L, N-Z-Amine AS 75g/L, MgSO4.7H2O 20g/L, citric acid 3g/L, KH2PO4 6.75g/L, (NH4)2SO4 5g/L, Na2HPO4.12H2O 3g/L, MgCl2 5g/L, NH4Cl 0.1g/L. When the pH rose to a value greater than the set point (pH 7.0) by 0.1U due to the depletion of glucose, the nutrient feeding solution was automatically added to increase the glucose concentration in the culture broth. Expression of the igf-2 gene was induced by adding IPTG to a final concentration of 0.06 mM. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4.2H2O, 1.8 mM KH2PO4; pH 7.3 |
| Solubilization Buffer | 140 mM NaCl, 2.7 mM KCl, 3M urea, 10 mM Na2HPO4.2H2O, 1.8 mM KH2PO4; pH 7.3 |
| Refolding Buffer | PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4.2H2O, 1.8 mM KH2PO4; pH 7.3 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.3 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 36h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were harvested from 22.5 ml of culture by centrifugation at 6000g for 15 min at 4degC and washed twice in PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4.2H2O, 1.8 mM KH2PO4; pH 7.3), then centrifuged again. The washed cell pellets were resuspended thoroughly in PBS buffer and disrupted by sonication using a sonicator (Model XL 2020, 419A microtipprobe, Misonix Inc., NY, USA) at a setting of 3. The sonicator was programmed to provide 10 s pulses with 5 s pause for a total period of 30 min. The disrupting pellets were washed with 50 ml PBS buffer containing 3 M urea to remove cell debris contaminants and were further centrifuged at 17,500xg for 30 min to isolate the inclusion bodies. The inclusion bodies were completely soluble in 50 ml PBS buffer (pH 12) containing 5 M urea at room temperature and the solubilized solution was clarified by centrifugation at 17,500xg for 30 min at 4degC. Refolding was initiated by diluting the solubilized protein in PBS buffer and the solution was then incubated at 4degC for 36 h. The refolded protein was centrifuged at 17,500xg for 30 min to remove any insoluble material. The supernatant fraction was collated and concentration by centrifugation using a type YM3 ultrafiltration membrane (molecular weight cutoff, 3000; Millipore, MA, USA) and then loaded to bind with the Ni-NTA gel (Qiagen). The gel was washed with 30 mM imidazole and eluted with 250 mM imidazole. The purified IGF-2 protein solution was dialyzed against PBS buffer and stored at 4degC prepared for bioactivity assay. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 86mg/L |
| Purity | 90% |
| Notes | |