Refolding Record:
| Protein | |
|---|---|
| Protein Name | Fibroblast growth factor receptor |
| Abbreviated Name | FGFR |
| SCOP Family | I set domains |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P21802 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Ligand-binding domain aa.125-249 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 132 |
| Molecular Weight | 15087.0 |
| Pi | 8.90899 |
| Molecular Weight | 15087.0 |
| Disulphides | 2 |
| Full Sequence |
MNSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCP AGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMES VVPSDKGNYT CVVENEYGSI NHTYHLDVV
MDIGINSDPNSSSVDKLAAALEHHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hung K-W, Kumar TKS, Chi Y-H, Chiu I-M, Yu C (2004) Biochemical and Biophysical Research Com, 317, 253-258 |
| Project Aim | Structure-Function |
| 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 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pET20b(+) |
| Expression Protocol | Cells were grown in LB broth containing 100 micrograms/ml ampicillin. Protein expression was induced by 1mM IPTG when OD600 reached 0.4, and cells were incubated for 4h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.4 |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | n/a |
| Solubilization Buffer | 20mM phosphate pH 7.5, 300mM NaCl, 8M Urea |
| Refolding Buffer | 20mM phosphate pH 7.5, 300mM NaCl, 50mM ammoniuim sulfate |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cells were harvested and lysed by French press. The inclusion bodies were issolved in solubilization buffer at room temperature, then loaded onto Ni-NTA affinity columns. The bound protein was washed with 10 column volumes of refolding buffer containing 20mM imidazole, then eluted with refolding buffer containing 500mM imidazole. The refolded and eluted protein was then loaded onto a heparin sepharose column and washed with 20mM phopshate buffer (pH 6.5) containing 20mM NaCl and 50mM ammonium sulfate. The protein was then eluted with 1M NaCl. |
| Refolding Assay | 15N -1H chemical shifts (ppm) |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | 95% |
| Notes | |