Refolding Record:
| Protein | |
|---|---|
| Protein Name | Brain-derived neurotrophic factor |
| Abbreviated Name | BDNF |
| SCOP Family | Neurotrophin |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P23560 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 230 |
| Molecular Weight | 25766.3 |
| Pi | 8.98 |
| Molecular Weight | 25766.3 |
| Disulphides | 3 |
| Full Sequence |
AP MKEANIRGQG GLAYPGVRTH GTLESVNGPK
AGSRGLTSLA DTFEHVIEEL LDEDQKVRPN EENNKDADLY TSRVMLSSQV PLEPPLLFLL EEYKNYLDAA NMSMRVRRHS DPARRGELSV CDSISEWVTA ADKKTAVDMS GGTVTVLEKV PVSKGQLKQY FYETKCNPMG YTKEGCRGID KRHWNSQCRT TQSYVRALTM DSKKRIGWRF IRIDTSCVCT LTIKRGR
|
| Notes | approximated sequence only |
| Expression | |
|---|---|
| Report | Inoue M, Akimaru J, Nishikawa T, Seki N, Yamada H. (1998) Biotechnol. Appl. Biochem., 28, 207-213 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | LC137 |
| Expression Temp | 38.0 |
| Expression Time | 3-20h |
| Expression Vector | pBR322 |
| Expression Protocol | no details provided |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 8M urea, 0.5M Tris-HCl, 1mM EDTA, 40mM DTT, 125mM trimethylammoniopropyl methanethiosulphonate pH 8.5 |
| Refolding Buffer | 50 mM Tris-HCl, 2 M urea, 0.7 mM oxidized glutathione, 0.1 mM DTT, 5% glycerol |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 9.8 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | GSSG/DTT |
| Redox Agent Concentration | 0.7mM/0,0.7mM/01mM |
| Refolding Protocol | During the solubilization process the protein was treated with trimethylammoniopropyl methanethiosulphonate which reversibly derivatizes free cysteine residues and prevents intermolecular interaction. Solubilized protein was added drop by drop to the refolding medium containing 2 M urea, 0.7 mM oxidized glutathione, 0.1 mM DTT, 5% glycerol in 50 mM Tris-HCl buffer, pH 9.8, and incubated at 4°C. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 5% |
| Refolding Yield | 50% |
| Purity | |
| Notes | |