Refolding Record:
| Protein | |
|---|---|
| Protein Name | Ribonuclease A |
| Abbreviated Name | RNase A |
| SCOP Family | Ribonuclease A-like |
| Structure Notes | |
| Organism | Yeast (Saccharomyces cerevisiae) |
| UniProt Accession | P53942 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 307 |
| Molecular Weight | 34875.0 |
| Pi | 8.99 |
| Molecular Weight | 34875.0 |
| Disulphides | Unknown |
| Full Sequence |
MVPPTVEASLESPYTKSYFSPVPSALLEQNDSPIIMGIDEAGRGPVLGPMVYAVAYSTQKYQDETIIPNYEFDDSKKLTDPIRRMLFSKIYQDNEELTQIGYATTCITPLDISRGMSKFPPTRNYNLNEQAHDVTMALIDGVIKQNVKLSHVYVDTVGPPASYQKKLEQRFPGVKFTVAKKADSLYCMVSVASVVAKVTRDILVESLKRDPDEILGSGYPSDPKTVAWLKRNQTSLMGWPANMVRFSWQTCQTLLDDASKNSIPIKWEEQYMDSRKNAAQKTKQLQLQMVAKPVRRKRLRTLDNWYR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hagen AJ, Hatton TA, Wang DI (1990) Biotechnol Bioeng, 35, 955-965 |
| 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 | Reversed Micelles |
| Wash Buffer | 6 M GuHCl in 0.025 M phosphate buffer, pH 7.5 + B-Mercaptoethanol |
| Solubilization Buffer | 1 M GuHCl + 1 mM EDTA in 0.1 M Tris, pH 8.7 |
| Refolding Buffer | 4-5 denatured RNase solution + 400 mM sodium dioctyl sulfosuccinate (AOT) in iso-octane |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.7 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 15 min |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | n/a,n/a,n/a,n/a |
| Refolding Protocol | Transfer of Denatured RNase to Reversed Micelles Equal volumes (typically 4 – 5 mL) of denatured RNase solution (containing 0.5 – 3 mg/mL RNase and 0.5 – 1 M GuHCl) and 400 mM AOT in iso-octane were combined in a 20-mL scintillation vial and stirred magnetically for 15 min at room temperature. The cloudy mixture was transferred to a capped centrifuge tube and centrifuged for 15 min, resulting in the formation of two clear phases, which were separated by careful pipetting. A blank sample containing no protein was treated in parallel. A thin layer of white precipitate was sometimes seen at the interface and was discarded. This precipitate was presumably the surfactant since very little protein was lost and it was also observed in the absence of protein. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,sodium dioctyl sulfosuccinate (AOT) |
| Additives Concentration | 400 mM |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |