Refolding Record:
| Protein | |
|---|---|
| Protein Name | Erythropoietin Receptor |
| Abbreviated Name | EPOR |
| SCOP Family | Fibronectin type III |
| Structure Notes | |
| Organism | Xenopus laevis |
| UniProt Accession | Q4W815 |
| 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 | 501 |
| Molecular Weight | 56211.5 |
| Pi | 5.27505 |
| Molecular Weight | 56211.5 |
| Disulphides | 0 |
| Full Sequence |
EDPTMTPEFLRHISEKIPEEYQNPHCFT
RDLNDFICFWEGERRKNASFSYSEDDQIKWCQLRTEVASNNTWWYICEFPVTDVVLFAGI
TISAYPCHKCQTAREIYINELVLLNPPLNVTVKEKQDPRGLLISWKPPHFQKNHDINNEI
KYQVNYSTPGADMQTVEVEAGNTEIFLTDIVPAAYTVTVRCKADGVSYNGYWSDWTAPIT
IATIIDLRLLLLLSIAIFVALIAGVGVYIFMRHGMYLKHKVWPQVPTPENNFQGLFTTHK
GNFKLWLGQADAYLLWISRHVFQEDPSSTLEVLSELPPAALPQSFNPNPLKDSYVVLDEN
RMPCSLEWLEAQRHKTVIVGAESMDSRLQTVNKDVVLEDTSKGQIAVKANNRVHSLEGDG
SQGEAFREDEYVEAPRMEHERHRVSRENSVSSDGKQSIPSSFEYTELQTCEGLLSPKPRP
VPPRMPLKYAYLDMSSSGEHSPPPSPNFYQNSPITNFLAPIYSQS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Aizawa, Y., Nogawa, N., Kosaka, N., Maeda, Y., Watanabe, T., Miyazaki, H., Kato, T. (2005) J Biochem (Tokyo), 138, 167-175 |
| Project Aim | Functional Studies |
| Fusion | N-terminal GST |
| 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 | 0.0 |
| Expression Vector | pGEX-6P-1 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M urea, 5 mM EDTA and 10 mM DTT in DPBS |
| Refolding Buffer | DTT and urea removed (no futher details given) |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 37.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Induction by IPTG allowed the expression of the insoluble GST and xlEPOR fusion protein in E. coli. After extensive washing, solubilization buffer was then added to the protein. The soluble fraction was then obtained using centrifugation and was subjected to step wise dialysis to allow refolding. Once refolded the protein was purified by application to a Glutathione Sepharose 4B column. The GST tag was then removed via digestion with PreScission protein protease, before being removed by dialysis and reapplication to the column. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | Extracellular domain of xlEPOR after cleavage of GSP tag MGAPSSLLFSTAHWRTVPFLLAFWVLLSTGTAEDPTMTPEFLRH ISEKIPEEYQNPHCFTRDLNDFICFWEGERRKNASFSYSEDDQIKWCQLRTEVASNNT WWYICEFPVTDVVLFAGITISAYPCHKCQTAREIYINELVLLNPPLNVTVKEKQDPRG LLISWKPPHFQKNHDINNEIKYQVNYSTPGADMQTVEVEAGNTEIFLTDIVPAAYTVT VRCKADGVSYNGYWSDWTAPITIATIIDLRLLLLLSIAIFVALIAGVGVYIFMRHGMY LKHKVWPQVPTPENNFQGLFTTHKGNFKLWLGQADAYLLWISRHVFQEDPSSTLEVLS ELPPAALPQSFNPNPLKDSYVVLDENRMPCSLEWLEAQRHKTVIVGAESMDSRLQTVN KDVVLEDTSKGQIAVKANNRVHSLEGDGSQGEAFREDEYVEAPRMEHERHRVSRENSV SSDGKQSIPSSFEYTELQTCEGLLSPKPRPVPPRMPLKYAYLDMSSSGEHSPPPSPNF YQNSPITNFLAPIYSQS Number of amino acids: 525 Molecular weight: 59672.6 Theoretical pI: 5.38 |