Refolding Record:
| Protein | |
|---|---|
| Protein Name | Somatotropin also known as Growth hormone |
| Abbreviated Name | GH |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Siganus guttatus (Rabbitfish) |
| UniProt Accession | Q9IBE5 |
| 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 | 179 |
| Molecular Weight | 20443.1 |
| Pi | 6.11 |
| Molecular Weight | 20443.1 |
| Disulphides | 2 |
| Full Sequence |
QPMTDSQRFSIAVSRIHYLHQVAQRSFFTFESSLSAEDQRQLNKIFLQDSCNSDYIRSPIDKHETQRSSVMKLLSISYRLVESWEYPSRALIGGSTNQISNKLSELKLGIRLLMEANQDGAEIFPESSAFQLDYQSLGTDDPRQMYELLACFKKDMHKVETYLTVAKCRLSPEANCTL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Funkenstein B, Dyman A, Lapidot Z, de Jesus-Ayson EG, Gertlerc A, Aysonb F (2005) Aquaculture, 250, 504 – 515 |
| 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(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pET-3d |
| Expression Protocol | For large-scale preparation, BL21(DE3) E. coli clone #2 was grown in 20 * 500 ml LB in the presence of ampicillin (100 Ag/ml) at 37 8C in 2-L flasks to an of OD600 0.8. Then, IPTG was added to 0.4 mM. The cells were grown for an additional 4 h, pelleted for 5 min at 10,000 *g in a Sorvall RC5C centrifuge at 4 8C and stored at - 20C |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.8 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | distilled water containing 0.1% Triton X-100 |
| Solubilization Buffer | 4.5 M urea buffered with 40 mM Trisma base in a total volume of 14 ml |
| Refolding Buffer | 10 mM Tris, pH 8, 0.2 mM cystein |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 0.2 mM |
| Refolding Protocol | In order to optimize refolding conditions, the IB pellet obtained from 2 L fermentation culture (20 ml) was divided into 4 aliquots. Each of the aliquots was solubilized in 4.5 M urea buffered with 40 mM Trisma base in a total volume of 14 ml. The pH was adjusted to 11.3 with 2–3 drops of 2 N NaOH, resulting in clearing the solution and then cysteine was added to 0.1 mM, 0.2 mM, 1 mM and 5 mM. The solution was stirred at 4 8C for 1 h, diluted with 2 volumes of cold water and dialyzed for 48 h against 5 -2 L of 10 mM Tris, pH 9 and applied to a Q-Sepharose (fast flow) (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) column 0.8 * 4 cm, pre-equilibrated with the same buffer at 4C. Elution was carried out using a discontinuous NaCl gradient in the same buffer and 1-ml fractions were collected. In these experiments most of the rfGH eluted with 100 mM NaCl. In subsequent refolding experiments the protein was refolded in the presence of 0.2 mM cysteine but dialyzed against 10 mM Tris, pH 8. The elution was carried out by discontinuous NaCl gradient and the rfGH was eluted with 50 mM NaCl. Protein concentration was monitored at 280 nm and monomer content was determined by gel filtration chromatography on a Superdex 75 column (1 *30 cm) Pharmacia LKB Biotechnology AB). In addition, aliquots of selected fractions were analyzed by SDS–PAGE (see below). |
| Refolding Assay | Western Blot,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | ~ 5 mg |
| Purity | 98% |
| Notes | Refolding Temperature not stated |