Refolding Record:
| Protein | |
|---|---|
| Protein Name | Neurotrophin-4/Neurotrophin-5 |
| Abbreviated Name | NT4/NT5 |
| SCOP Family | Neurotrophin |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P34130 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 130 |
| Molecular Weight | 13923.6 |
| Pi | 8.90055 |
| Molecular Weight | 13923.6 |
| Disulphides | 3 |
| Full Sequence |
GVSETAPASR RGELAVCDAV SGWVTDRRTA VDLRGREVEV LGEVPAAGGS PLRQYFFETR CKADNAEEGG PGAGGGGCRG VDRRHWVSEC KAKQSYVRAL TADAQGRVGW RWIRIDTACV
CTLLSRTGRA
|
| Notes | Full mature protein (without pre-pro sequence) expressed and refolded. |
| Expression | |
|---|---|
| Report | Fandl JP, Tobkes NJ, McDonald NQ, Hendrickson WA, Ryan TE, Nigam S, Acheson A, Cudny H, Panayotatos N (1994) J Biol Chem, 269, 755-759 |
| Project Aim | Crystallography |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | RFJ42 |
| Expression Temp | 37.0 |
| Expression Time | overnight |
| Expression Vector | pRG227 |
| Expression Protocol | Cells were grown to late log phase in LB medium at 37degC, then induced with 1mM IPTG and grown overnight. |
| 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 | Dilution |
| Wash Buffer | not stated |
| Solubilization Buffer | 6M GdnHCl, 50mM TrisHCl pH 8.5, 5mM EDTA |
| Refolding Buffer | not stated |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were harvested, washed and lysed in 100mM TrisHCl pH 8.0, 50mM EDTA. Inclusion bodies were collected by centrifugation and dissolved in solubilization buffer. The protein was refolded under denaturing redox conditions and purified by cation exchange chromatography followed by gel filtration |
| Refolding Assay | Amino acid sequencing |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | 95%+ |
| Notes | |