Refolding Record:
| Protein | |
|---|---|
| Protein Name | Erythropoietin Receptor |
| Abbreviated Name | EPOR |
| SCOP Family | Fibronectin type III |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P19235 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 228 |
| Molecular Weight | 25001.4 |
| Pi | 5.29758 |
| Molecular Weight | 25001.4 |
| Disulphides | 2 |
| Full Sequence |
PPPNL PDPKFESKAA LLAARGPEEL LCFTERLEDL VCFWEEAASA GVGPGNYSFS YQLEDEPWKL CRLHQAPTAR
GAVRFWCSLP TADTSSFVPL ELRVTAASGA PRYHRVIHIN EVVLLDAPVG LVARLADESG HVVLRWLPPP ETPMTSHIRY EVDVSAGNGA GSVQRVEILE GRTECVLSNL RGRTRYTFAV RARMAEPSFG GFWSAWSEPV SLLTPSDLDP SGC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Johnson DL, Middleton SA, McMahon F, Barbone FP, Kroon D, Tsao E, Lee WH, Mulcahy LS, Jolliffe LK (1996) Protein Expression and Purification, 7, 104-113 |
| Project Aim | Recombinant Protein Expression |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | overnight |
| Expression Vector | pET11a |
| Expression Protocol | Enriched M9ZB media containing (per L) 20g N-Z-Amine A, 5g NaCl, 10g M9 minimal salts, 0.12g MgSO4, 0.2g glucose, 100mg ampicillin and 25mg chloramphenicol was equilibrated in a fermentor at 37degC with 20% dissolved oxygen at pH 6.8. The medium was inoculated with 50ml trsformed E.coli glycerole stock. When OD600 reached 25.0, expression was induced by the addition of 1mM IPTG and the pH reduced to 6.1 with HCl and the cells were incubated overnight. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 25 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | water |
| Solubilization Buffer | 100mM TrisHCl pH 8.5, 3.5M urea, 10mM lylsine, 10mM DTT |
| Refolding Buffer | 50mM TrisHCl pH 8.5, 3mM EDTA, 10mM lysine, 2mM GSH, 0.2mM GSSG |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 6 days |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2mM/0.2mM,2mM/0.2mM |
| Refolding Protocol | 74.3g cell paste was lysted in 300ml of 10mM TrisHCl pH 7.5, 150mM NaCl with 51mg PMSF, 600mg lysozyme, 1mM MgCl2, 12500 units Benzonase. The mixture was incubated at room temperature for 1.5h with occasional agitation, then centrifuged (12000g, 15min, 4degC). The pellet was resuspended in 10mM TrisHCl pH 7.5, 1mM EDTA, 3% NP-40 and then sonicated for 1min. The suspension was then centrifuged (8000g, 15min, 4degC) and the pellet was washed with 300ml water. The pellet was then resuspended by sonication (20sec) and centrifuged (12000g, 15min, 4degC). The insoluble pellet was then resuspended in 9ml solubilization buffer per gram of protein and centrifuged (12000g, 15min, 4degC). The OD280 of the solubilized protein was determined and 30ml of the solubilized protein was diluted to 1020ml in refolding buffer and stored at 4degC for 6 days. |
| Refolding Assay | Bioactivity,Ligand Binding,Gel filtration chromatography,Amino acid sequencing,Mass spectrometry |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |