Refolding Record:
| Protein | |
|---|---|
| Protein Name | Insulin-like growth factor II |
| Abbreviated Name | IGF2 |
| SCOP Family | Insulin-like |
| Structure Notes | |
| Organism | Oncorhynchus mykiss |
| UniProt Accession | Q02816 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 70 |
| Molecular Weight | 7871.8 |
| Pi | 5.1 |
| Molecular Weight | 7871.8 |
| Disulphides | 3 |
| Full Sequence |
EVASAETLCGGELVDALQFVCEDRGFYFSRPTSRSNSRRSQNRGIVEECCFRSCDLNLLEQYCAKPAKSE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Gentil V, Martin P, Smal J, Le Bail PY. (1996) General and Comparative Endocrinology, 104, 156-167 |
| Project Aim | Over expression & Renaturation |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | overnight |
| Expression Vector | parHS3 |
| Expression Protocol | Expression of rtIGF-II and Isolation of Inclusion Bodies Individual selected colonies were used to inoculate 500 ml of Luria–Bertani medium containing 50 µg/ml ampicillin (Sambrook et al., 1989). After 4 hr of growth at 37° with vigorous shaking, the culture was used to inoculate a fermentor (25 liters of Luria–Bertani medium containing 50 µg/l ampicillin). The bacteria were grown at 37° with shaking until the absorbency at 600 nm was near 0.9. The expression was then induced by adding IPTG to a final concentration of 1 mM and the cells were incubated overnight at 37°. Cells were concentrated to 2 liters of culture and centrifuged at 3000g for 15 min at 4°. The pellet was resuspended in two volumes of 50 mM Tris–HCl, 2 mM EDTA at pH 8.0 and Nonidet-P40 0.5%. The inclusion bodies were collected by centrifugation and washed three times with the same solution. The wet pellet was stored at 220° until use. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.9 = 600 |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50 mM Tris–HCl, 2 mM EDTA at pH 8.0 and Nonidet-P40 0.5% |
| Solubilization Buffer | 5 ml 8 M urea, B-mercaptoethanol 2%, pH 9.5 |
| Refolding Buffer | 20 mM ammonium bicarbonate, pH 8.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 20.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Solubilization, Purification, and Refolding of rtIGF-II Protein Inclusion bodies (50 mg of wet pellet) containing the trout IGF-II peptide were solubilized by suspension in 5 ml 8 M urea, b-mercaptoethanol 2%, pH 9.5, at 20° for 15 min. The dissolved inclusion bodies were centrifuged for 15 min at 12,000g at 20°. The supernatant was filtered on glass microfiber filters (ref: 1820 025, What- man) and fractioned by denatured gel filtration chromatography on a TSK-HW55 (S) column (5 3 100 cm, Merck), which was equilibrated with 6 M urea, pH 9.5. The proteins were eluted at 30 ml/hr flow rate and 5-ml fractions were collected. After absorbency reading at 280 nm of the fractions, those corresponding to the molecular weight of IGF were kept. Refolding of the reduced and purified peptide was performed by dialysis using a membrane with a cutoff of 3.5 kDa (ref: FIG. 132 724, Spectrum Medical Industries, Inc.) with 20 mM ammonium bicarbonate, pH 8.5. The fraction was lyophilized and stored at -20°. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 820 ug/l |
| Purity | n/a |
| Notes | n/a |