Refolding Record:
| Protein | |
|---|---|
| Protein Name | Ribonuclease 2 |
| Abbreviated Name | RNase2 |
| SCOP Family | Ribonuclease A-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P10153 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 138 |
| Molecular Weight | 15900.1 |
| Pi | 9.2 |
| Molecular Weight | 15900.1 |
| Disulphides | Unknown |
| Full Sequence |
SLHVKPP QFTWAQWFET QHINMTSQQC TNAMQVINNY QRRCKNQNTF LLTTFANVVN VCGNPNMTCP SNKTRKNCHH
SGSQVPLIHC NLTTPSPQNI SNCRYAQTPA NMFYIVACDN RDQRRDPPQY PVVPVHLDRI I
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Sun L, Li M-S, Fisher M, Spry CJF (1995) Protein Expression and Purification, 6, 685-692 |
| Project Aim | Functional Studies |
| Fusion | N-terminal maltose binding protein |
| Protein Expression and Production | Protein expressed and purified in native conformation prior to denaturation and refolding. |
| Expression Host | None |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 0 |
| Expression Vector | |
| Expression Protocol | |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 10M urea, 10mM DTT, 50mM TrisHCl pH 8.2, 1.0mM EDTA |
| Refolding Buffer | 0.1M TrisHCl pH 8.2, 0.1mM EDTA, 1.0mM GSH |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 8.2 |
| Refolding Temperature | 22.0 |
| Protein Concentration | 20microg/ml |
| Refolding Time | 4h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1.0mM/0.5mM |
| Refolding Protocol | The protein was expressed and purified solubly as an MBP-fusion protein, the MBP fusion partner was then cleaved. 8microg of the cleaved protein was then denatured in solubilization buffer for 30min at 37degC. The solution was then diluted 100-fold to a final concentration of 20microg/ml with refolding buffer and preincubated at 0degC for 5min. Reoxidation was initiated by the addition of 0.025vol of oxidized glutathione to a final concentration of 0.5mM and the mixture was then incubated for 4h at 22degC |
| Refolding Assay | Enzyme activity,Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 11% |
| Purity | n/a |
| Notes | n/a |