Refolding Record:
| Protein | |
|---|---|
| Protein Name | Ribonuclease A |
| Abbreviated Name | RNase A |
| SCOP Family | Ribonuclease A-like |
| Structure Notes | |
| Organism | Bovine |
| UniProt Accession | P61823 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 124 |
| Molecular Weight | 13690.3 |
| Pi | 8.63544 |
| Molecular Weight | 13690.3 |
| Disulphides | 4 |
| Full Sequence |
KETA AAKFERQHMD SSTSAASSSN YCNQMMKSRN LTKDRCKPVN TFVHESLADV QAVCSQKNVA CKNGQTNCYQ SYSTMSITDC RETGSSKYPN CAYKTTQANK HIIVACEGNP YVPVHFDASV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Sakono M, Ichinose H, Goto M (2003) J Biosci Bioeng, 96, 275-278 |
| Project Aim | Protein refolding |
| Fusion | None |
| 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 | Chaperone-assisted/reversed micelles |
| Wash Buffer | n/a |
| Solubilization Buffer | TrisHCl, 8M urea, 30mM 2-mercaptoethatnol pH 8.5 |
| Refolding Buffer | 0.01M TrisHCl pH 8.5, 1mM ATP, 1mM MgCl2, 1mM KCL, 2g/L GroEL |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 30.0 |
| Protein Concentration | n/a |
| Refolding Time | 6h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 4mM/0.4mM,4mM/0.4mM,4mM/0.4mM |
| Refolding Protocol | The protein was dissolved in 5ml solubilization buffer and incubated for 24h at room temperature. The denatured protein was then precipiated with ice-cold acetone, harvested by centrifugation (6000g) and lyophilized for 24h. The protein was then solubilized into a reversed micellar solution - this solution was prepared by injecting 0.216-0.432ml of refolding buffer into isooctane containing 400mM AOT so that the total volume was 1ml. The RNase was solubilized by ultra sonication for 10min, then the revesred micellar solution was incubated for 50min at 30degC. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | GroEL |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 90% |
| Purity | n/a |
| Notes | 90% refolding recovery with GSH/GSSG, 60% without. |