Refolding Record:
| Protein | |
|---|---|
| Protein Name | Tumor necrosis factor alpha |
| Abbreviated Name | TNF-alpha |
| SCOP Family | TNF-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01375 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Trimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 233 |
| Molecular Weight | 25644.4 |
| Pi | 6.43641 |
| Molecular Weight | 25644.4 |
| Disulphides | 1 |
| Full Sequence |
MSTESMIRDV ELAEEALPKK TGGPQGSRRC LFLSLFSFLI VAGATTLFCL LHFGVIGPQR EEFPRDLSLI SPLAQAVRSS SRTPSDKPVA HVVANPQAEG QLQWLNRRAN ALLANGVELR DNQLVVPSEG LYLIYSQVLF KGQGCPSTHV LLTHTISRIA VSYQTKVNLL SAIKSPCQRE TPEGAEAKPW YEPIYLGGVF
QLEKGDRLSA EINRPDYLDF AESGQVYFGI IAL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Davis JM, Narachi MA, Alton NK, Arakawa T (1987) Biochemistry, 26, 1322-1326 |
| Project Aim | Biophysical Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | C600 |
| Expression Temp | 37.0 |
| Expression Time | n/a |
| Expression Vector | not stated |
| Expression Protocol | Protein was expressed by standard fermentation processes. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | high concentration urea, DTT |
| Refolding Buffer | 40mM TrisHCl, pH 8.5 |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Harvested cells were disrupted with a homogenizer and then centrifuged - protein was present in both soluble and insoluble fractions, although more in the latter. The pelleted protein was solublized in concentrated urea in the presence of DTT and purified in the presence of urea and DTT, except for the last step in which DTT was removed. The protein was purified firstly bu cation-exchange chromatography at pH 4.5 followed by anion-exchange chromatography at pH 9.0. The purified protein was diluted into 10 volumes of refolding buffer and then concentrated to ~2mg/ml. The protein was then loaded onto a Sephadex G-75 column equilibrated with 40mM TrisHCl, 0.1M NaCl pH 8.5. |
| Refolding Assay | Far-UV Circular Dichroism |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |