Refolding Record:
| Protein | |
|---|---|
| Protein Name | Prolactin receptor extracellular domain |
| Abbreviated Name | Prolactin receptor ECD |
| SCOP Family | Fibronectin type III |
| Structure Notes | |
| Organism | Rat |
| UniProt Accession | P05710 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Extracellular domain aa.24-229 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 207 |
| Molecular Weight | 24278.3 |
| Pi | 5.65615 |
| Molecular Weight | 24278.3 |
| Disulphides | 2 |
| Full Sequence |
MGKPEIHK CRSPDKETFT CWWNPGTDGG LPTNYSLTYS KEGEKTTYEC PDYKTSGPNS CFFSKQYTSI WKIYIITVNA
TNQMGSSSSD PLYVDVTYIV EPEPPRNLTL EVKQLKDKKT YLWVKWSPPT ITDVKTGWFT MEYEIRLKPE EAEEWEIHFT GHQTQFKVFD LYPGQKYLVQ TRCKPDHGYW SRWSQESSVE MPNDFTLKD
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Sandowski Y, Nagano M, Bignon C, Djiane J, Kelly PA, Gertler A (2005) Mol Cell Endocrinol, 115, 1-11 |
| 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 | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 12h |
| Expression Vector | pTrc99A |
| Expression Protocol | Cells were grown in TB media at 37degC, when A600 reached 0.9, 0.4mM IPTG was added and cells were grown for a further 12h then harvested by centrifugation (10min x 16000g). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.9 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | PBS, 25% (w/v) sucrose, 5mM EDTA, 1% (v/v) Triton X-100 |
| Solubilization Buffer | 4.5M urea, 100mM Tris |
| Refolding Buffer | 4.5M urea, 100mM 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 100ml cold water, frozen, thawed and sonicated (4x30s). The inclusion bodies wre collected by centrifugation (30min x 27000g) and resuspended by brief sonication in 50ml wash buffer. This process was repeated 3 more times. The pellet was then dissolved in 300ml solubilization buffer. The pH was increased to 11.3 with NaOH and cysteine was added to 1mM. The clear solution was stirred at 4degC for 1h, then diluted with 2 volumes of ice coled water before being dialyzed for 36h against 4 x 10L of 10mM TrisHCl pH 8.6. The solution was then loaded at 200ml/h onto a Q-sepharose column preequilibrated with the same buffer. The protein was eluted using a discontinuous NaCl gradient in the same buffer at 100ml/h and 5ml fractions were collected. |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | <10% |
| Purity | >98% |
| Notes | less than 10% of the expressed protein refolded to monomeric state |