Refolding Record:
| Protein | |
|---|---|
| Protein Name | Growth Hormone |
| Abbreviated Name | GH |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Carp (Cyprinus carpio) |
| UniProt Accession | P10298 |
| 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 | 210 |
| Molecular Weight | 23765.3 |
| Pi | 5.82288 |
| Molecular Weight | 23765.3 |
| Disulphides | 2 |
| Full Sequence |
MARVLVLLSV VLVSLLVNQG RASDNQRLFN NAVIRVQHLH QLAAKMINDF EDSLLPEERR QLSKIFPLSF CNSDYIEAPA GKDETQKSSM LKLLRISFHL IESWEFPSQS LSGTVSNSLT VGNPNQLTEK LADLKMGISV LIQACLDGQP NMDDNDSLPL PFEDFYLTMG ENNLRESFRL LACFKKDMHK VETYLRVANC
RRSLDSNCTL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Fine M, Sakal E, Vashdi D, Daniel V, Levanon A, Lipshitz O, Gertler A (1993) Gene, 89, 51-61 |
| Project Aim | Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | A1645 |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pBR322 |
| Expression Protocol | Cells were grown in L broth containing 100microg/ml ampicillin at 30degC. During the logarithmic growth phase, expression was induced by a temperature shift to 42degC. Cells were grown for a further 120min, harvested by centrifugation and suspended in 50mM potassium phosphate buffer (pH 7.8). |
| Method of Induction | Temperature Shift |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | distilled water |
| Solubilization Buffer | 4.5M urea, 10mM TrisHCl pH 8.0 |
| Refolding Buffer | 40mM TrisHCl, 4.5M urea, 0.1mM cysteine pH 11.3 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 11.3 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 48h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 0.1mM |
| Refolding Protocol | Cells (80g) stored at -80degC were thawed, homogenized and incubated for 30min in 10mM EDTA pH 8.0 in the presence of 0.5mg/ml lysozyme. The cells were sonicated and pelleted by centrifugation (30min, 25000g). The pellet was washed twice with distilled water by sonication, then reprecipitated and finally solubilized in 1.6L solubilization buffer. Insoluble material was removed, the concentration of TrisHCl was increased to 40mM, the pH was raised to 11.3 and cysteine was added at 0.1mM. After stirring for 48hr at 4degC, the solution was loaded onto a Q-sepharose column equilibrated with 20mM TrisHCl pH 8.0 at 4deg C. Elution was performed using a discontinuous NaCl gradient in the same buffer. |
| Refolding Assay | Gel filtration chromatography |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |