Refolding Record:
| Protein | |
|---|---|
| Protein Name | Prolactin receptor extracellular domain |
| Abbreviated Name | Prolactin receptor ECD |
| SCOP Family | Fibronectin type III |
| Structure Notes | |
| Organism | Rabbit (Oryctolagus cuniculus) |
| UniProt Accession | P14787 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Extracellular domain aa.29-235 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 207 |
| Molecular Weight | 24024.4 |
| Pi | 6.99888 |
| Molecular Weight | 24024.4 |
| Disulphides | 2 |
| Full Sequence |
MGK PFIFKCRSPE KETFTCWWRP GADGGLPTNY TLTYHKEGET ITHECPDYKT GGPNSCYFSK KHTSIWTIYI ITVNATNQMG SSVSDPRYVD VTYIVEPDPP VNLTLEVKHP EDRKPYLWVK
WLPPTLVDVR SGWLTLQYEI RLKPEKAAEW ETHFAGQQTQ FKILSLYPGQ KYLVQVRCKP DHGFWSVWSP ESSIQIPNDF TMKD
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Bignon C, Sakal E, Belair L, Chapnik-Cohen N, Djiane J, Gertler A (1994) J Biol Chem, 269, 3318-3324 |
| 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 | W5 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pTrc99A |
| Expression Protocol | Cells were grown in LB medium at 37degC until A600 reached 0.9, and were induced with 1mM IPTG and grown for a further 4h. Cells were harvested by centrifugtaion (10min x 16000g). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.96 |
| Cell Disruption Method | Osmotic shock + sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | distilled water |
| Solubilization Buffer | 4.5M urea, 40mM Tris |
| Refolding Buffer | 4.5M urea, 40mM Tris, 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 | 1h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 0.1mM |
| Refolding Protocol | The cell pellet was resuspended in cold 10mM EDTA, 10mM TrisHCl pH 8.0 in the presence of 0.5mg/ml lysozyme and incubated for 30min at 4degC with occasional mixing. Cells were then sonicated and centrifuged (30min x 25000g). The pellet was sonicated in distilled water, pelleted twice, then solubilized in 1.5L solubilization buffer. The pH was adjusted to 11.3 with NaOH, cysteine was added to 0.1mM and the cleared supernatant was stirred at 4degC (if the mixture was not all solubilized, the suspension was centrifuged prior to stirring). After 1hr of stirring, the solution was diluted with 2 volumes of ice-cold water and dialyzed for 36h against 2x 20L of 10mM Tris pH 8.6 at 4degC. The solution was then loaded at 200ml/h onto a Q-sepharose column preequilibrated with 10mM Tris pH 8.6 at 4degC. The protein was eluted using a discontinuous NaCl gradient in the same buffer at a rate of 100ml/h and 5ml fractions were collected. |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 1.8-2.5mg/L |
| Purity | >98% |
| Notes | Only extracellular domain purified Protein was ~70% insoluble Longer refolding periods (5-24h) decreased yield |