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 | Subtilisin digestion-based fragment aa.16-124 |
| Chimera | n/a |
| Variants | A122C |
| Chain Length | 109 |
| Molecular Weight | 11991.4 |
| Pi | 8.58685 |
| Molecular Weight | 11991.4 |
| Disulphides | 4 |
| Full Sequence |
STSAASSSNY CNQMMKSRNL TKDRCKPVNT FVHESLADVQ AVCSQKNVAC KNGQTNCYQS YSTMSITDCR ETGSSKYPNC AYKTTQANKH IIVACEGNPY VPVHFDCSV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Backer MV, Gaynutdinov TI, Aloise R, Przekop K, Backer JM (2002) Protein Expression and Purification, 26, 455-461 |
| 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 | Tuner(DE3)pLacI |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pET32a(+) |
| Expression Protocol | Cells were grown in CircleGrow medium (Q-Biogen), and protein expression was induced by 1mM IPTG when OD600 reached 0.4-0.5. Cells were grown for a further 4h at 37degC with shaking at 250-280rpm then harvested by centrifugation (15min, 5000g). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.4-0.5 |
| Cell Disruption Method | Chemical |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | BugBuster reagent (Novagen)/CelLyticB reagent (Sigma), 1x HisBind buffer (Novagen) |
| Solubilization Buffer | 8M urea, 20mM TrisHCl, 100mM NaCl pH 8.0 |
| Refolding Buffer | 1: 100mM TrisAcetate, 100mM NaCl, 0.5M arginine, 1mM GSH, 0.4mM GSSG pH 8.6/2: 100mM NaPi, 150mM NaCl pH 7.2 |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 8.6 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1mM/0.4mM |
| Refolding Protocol | The cells were lysed using BugBuster reagent (Novagen), then inclusion bodies were washed with BugBuster, CelLyticB reagent diluted 1:10 with water (Sigma) and 1x HisBind buffer (Novagen). Inclusion bodies were then dissolved in solubilization buffer with sonication, and the protein concentrations were determined by SDS-PAGE on 17.5% gels. Synthetic peptide, corresponding to aa.1-15 of bovine ribonuclease A was added to the solubilized inclusion bodies at a molar ratio or 1:1. The mixtures were then dialyzed against 20 volumes of refolding buffer 1 for 20h, followed by 24h against 100V of refolding buffer 2. Following dialysis, the protein was centrifuged at 25000g for 15min, mixed with 1/10 volume of ice-cold 10% TFA, incubated on ice for 5min and centrifuged again (15min x 25000g). The supernatants were then passed through a 1ml HiTrap Sepharose SP column and the column was washed with 20mM NaOAc, pH 6.5. The protein was then eluted in 20mM NaOAc, 1M NaCl pH 6.5. Selected fractions were then purified further by RP HPLC, eluted at 2ml/min with 0.1% TFA(v/v) and a linear gradient of acetonitril (0-65% over 30min) and collected in tubes containing one volume of ice cold 40mM NaOAc pH 6.5. The protein was then passed through a HiTrap Sepharose SP column again, then dialyzed for 24h against 100 volumes of 20mM NaOAc, 150mM NaCl pH 6.5, then centrifuged (15min x 25000g) |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 0.5M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |