Refolding Record:
| Protein | |
|---|---|
| Protein Name | Prolactin receptor extracellular domain |
| Abbreviated Name | Prolactin receptor ECD |
| SCOP Family | Fibronectin type III |
| Structure Notes | |
| Organism | Oncorhynchus mykiss |
| UniProt Accession | Q9DE08 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 210 |
| Molecular Weight | 24060.7 |
| Pi | 5.18511 |
| Molecular Weight | 24060.7 |
| Disulphides | 0 |
| Full Sequence |
ARHTPPGK PKLTSCRSPD KETFTCWWEA GSDGGRPTTY SLFYHKENSE TVYECPDYHT AGANSCFFNK NETSIWVNYN ITVVATNALG NNFSDPVDVD VVYIVQPHTP ENVTVGVLED EDGPFLRVSW EPPRKADTRS GWITLIYELR VKLEEAEEWE EHRAGQQKMF NNFSLHSGGV YMVQVRCKPD HGFWSEWSTA SYVKVPDYIP RE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Sandowski Y, Cohen Y, Le Rouzic P, Bignon C, Rentier-Delrue F, Djiane J, Prunet P, Gertler A (2000) General and Comparative Endocrinology, 118, 302-309 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | MON105 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pMON3403 |
| Expression Protocol | Cells were incubated in TB medium with shaking at 37degC until A600 reached 0.9. 0.05mg/ml nalidixic acid was added to induce expression, cells were incubated for an additional 4h and then harvested by centrifugation (5min, 5000g) and frozen at -20degC. |
| Method of Induction | Nalidixic Acid |
| Cell Density at Induction | OD 600 = 0.9 |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | n/a |
| Solubilization Buffer | 4.5M urea, 10mM Tris |
| Refolding Buffer | 10mM TrisHCl pH 8.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 1h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 1mM |
| Refolding Protocol | The inclusion body pellet from 5L culture was resuspended in 600ml solubilization buffer. The pH was increased to 11.3 with NaOH and cysteine was added to 1mM. The solution was stirred at 4degC for 1h, diluted with 2 volumes of cold water and dialyzed for 48h against 4x 10L refolding buffer. The solution was then loaded at 120ml/h on a Q sepharose column and th protein was purified by ion exchange chromatography |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |