Refolding Record:
| Protein | |
|---|---|
| Protein Name | Ribonuclease A |
| Abbreviated Name | RNase A |
| SCOP Family | Ribonuclease A-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P07998 |
| 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 | 111 |
| Molecular Weight | 12502.1 |
| Pi | 8.67 |
| Molecular Weight | 12502.1 |
| Disulphides | 4 |
| Full Sequence |
MSAASS STYCNQMMRR RNMTQGRCKP VNTFVHEPLV DVQNVCFQEK VTCKNGQGNC YKSNSSMHIT DCRLTNGSRY PNCAYRTSPK ERHIIVACEG SPYVPVHFDA SVEDS
|
| Notes | with P19A and S20A mutations to increase protein yield |
| Expression | |
|---|---|
| Report | Backer MV, Gaynutdinov TI, Gorshkova II, Crouch RJ, Hu T, Aloise R, Arab M, Przekop K, Backer JM (2003) J Controlled Release, 89, 499-511 |
| Project Aim | Drug Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | OrigamiB(DE3)pLacI |
| Expression Temp | 37.0 |
| Expression Time | 12-16h |
| Expression Vector | pET29a(+) |
| Expression Protocol | Cells were grown in Circle Grow medium (Q-Biogen), expression was induced with 1mM IPTG when OD600 reached 0.5-0.7. Cells were grown overnight at 37degC then harvested by centrifugation (15min x 5000g). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5-0.7 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | not stated |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | BugBuster (Novagen)/CelLytic B (Sigma), HisBind buffer (Novagen) |
| Solubilization Buffer | 20mM TrisHCl, 100mM NaCl, 8M Urea pH 8.0 |
| Refolding Buffer | 100mM Tris-Acetate, 100mM NaCl, 0.5M arginine, 1mM GSH, 0.4mM GSSG pH 8.6 |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 8.6 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1mM/0.4mM,1mM/0.4mM |
| Refolding Protocol | The cells were lysed using BugBuster reagent according to the manufacturer instructions of the manufacturer. The inclusion bodies were were centrifuged and washed with BugBuster reagent, followed by CelLytic B reagent (Sigma) diluted 1:10 with water, and then finally 1x HisBind buffer (Novagen). The concentration of protein was determined by RP HPLC. Synthetic peptide (aa.18-125 or was aa.18-127) was added to the solubilized inclusion bodies at a molar ratio of 2:1 peptide:protein. The protein/peptide mixture was then refolded in refolding buffer and then purified using ion-exchange chromatography and RP HPLC. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 0.5M |
| Refolding Yield | |
| Purity | |
| Notes | |