Refolding Record:
| Protein | |
|---|---|
| Protein Name | Fel d 1 |
| Abbreviated Name | Fel d 1 |
| SCOP Family | Uteroglobin like |
| Structure Notes | |
| Organism | Felis domesticus (cat) |
| UniProt Accession | P30438 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Heterodimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | Chain 1 and Chain 2 joined by linker |
| Variants | n/a |
| Chain Length | 168 |
| Molecular Weight | 18795.6 |
| Pi | 5.08418 |
| Molecular Weight | 18795.6 |
| Disulphides | 3 |
| Full Sequence |
VKM AETCPIFYDV FFAVANGNEL LLDLSLTKVN ATEPERTAMK KIQDCYVENG LISRVLDGLV MTTISSSKDC MGEAVQNTVE
DLKLNTLGR EICPAVKR DVDLFLTGTP DEYVEQVAQY KALPVVLENA RILKNCVDAK MTEEDKENAL SVLDKIYTSP LC HHHHHH
|
| Notes | UniProt ID of Chain 2: FEL1B_FELCA |
| Expression | |
|---|---|
| Report | Kaiser L, Gronlund H, Sandalover T, Ljunggren H, Schneider G, van Hag-Hamsten M, Achour A (2003) Acta Crystallographica Section D, 559, 1103-1105 |
| Project Aim | Crystallography |
| Fusion | C-terminal hexahistidine tag |
| 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 | not stated |
| Expression Vector | pET20b |
| Expression Protocol | Protein expression was induced with IPTG (no further details provided). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | 20mM TrisHCl, 6M Urea, 0.5M NaCl, 20mM imidazole pH 8.0 |
| Solubilization Buffer | 20mM TrisHCl, 6M GdnHCl, 0.5M NaCl, 5mM imidazole pH 8.0 |
| Refolding Buffer | 20mM TrisHCl, 0.5M NaCl, 20mM imidazole pH 8.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a,n/a |
| Refolding Protocol | The isolated inclusion bodies were dissolved in solubilization buffer and bound to a Hi-Trap Ni-NTA column. The column buffer was changed to wash buffer, and then a linear 12 column volume gradient was used with wash buffer and refolding buffer. The refolded protein was further purified by size exclusion and MonoQ ion-exchange chromatography. |
| Refolding Assay | Gel filtration chromatography |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 10mg/L |
| Purity | |
| Notes | |