Refolding Record:
| Protein | |
|---|---|
| Protein Name | Peroxidase C1C |
| Abbreviated Name | PRXC1C |
| SCOP Family | CCP-like |
| Structure Notes | |
| Organism | Armoracia rusticana (Horseradish) (Armoracia lapha |
| UniProt Accession | P15233 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 324 |
| Molecular Weight | 35589.2 |
| Pi | 6.12 |
| Molecular Weight | 35589.2 |
| Disulphides | 4 |
| Full Sequence |
QLTPTFYDNSCPNVSNIVRDIIINELRSDPSIAASILRLHFHDCFVNGCDA
SILLDNTTSFRTEKDAFGNANSARGFPVVDRIKAAVERACPRTVSCADVLTIAAQQSVNLAGGPSWRVPLGRRDSRQAFLDLANANLPAPSFTLPELKAAFANVGLNRPSDLVALSGGHTFGKNQCRFIMDRLYNFSNTGLPDPTLNTTYLQTLRQQCPRNGNQSVLVDFDLRTPTVFDNKYYVNLKEQKGLIQSDQELFSSPNATDTIPLVRSYADGTQTFFNAFVEAMNRMGNITPLTGTQGEIRLNCRVVNSNSLLHDIVEVVDFVSSM
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Gazaryan IG, Doseeva VV, Galkin AG, Tishkov VI. (1994) FEBS Letters, 354, 248-250 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | Jm109 |
| Expression Temp | 30.0 |
| Expression Time | 00 |
| Expression Vector | pSA261 |
| Expression Protocol | The recombinant enzyme was produced according to the previously described procedure [2] with the following modifications. E. coli JM 1091 pSA261 was grown using 300 ml LB medium in 10 mM Tris-HCl, pH 8.0, with ampicillin (lOO&ml) and 0.4% glycerol at 30°C. Gene expression was induced by 0.2 mM IPTG added in the middle of the log-phase of cell growth. The cells were harvested and son&ted (22 kHz, 10 min) in the presence of 2 M NaCl and 10 mM DTT. The disrupted mixture was incubated for 1.5 h and then the sonication procedure was repeated. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 0.05 M Tris-HCl buffer, pH 8.5 |
| Solubilization Buffer | 10 ml of 6 M urea containing 1 mM DTT |
| Refolding Buffer | 2 M urea, 0.7 mM oxidized glutathione, 0.1 mM D’IT, 5 mM calcium chloride, 5% glycerol in 50 mM Tris-HCl buffer, pH 9.8 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 9.8 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | GSSG/DTT |
| Redox Agent Concentration | 0.7/0.1 mM |
| Refolding Protocol | The supernatant was removed and the pellet was washed with 0.05 M Tris-HCl buffer, pH 8.5, and solubilized in 10 ml of 6 M urea containing 1 mM DTT. The solubilized protein (95% purity, 2 mg/ml) was added drop by drop to the refolding medium (2 1) containing 2 M urea, 0.7 mM oxidized glutathione, 0.1 mM D’IT, 5 mM calcium chloride, 5% glycerol in 50 mM Tris-HCl buffer, pH 9.8, and incubated at 4°C. 5 PM hemin was added after the overnight incubation. The activity was measured and the active enzyme was precipitated by ammonium sulfate (60% of saturation) after the refolding was completed. The pellet was dissolved in 30 ml water and applied in 15-ml portions to a column (5.2 x 80 cm) with Toyopearl HW 55F equilibrated with 0.05 M Tris-HCl, pH 7.0, containing 0.1 M NaCl. Active fractions were collected and stored frozen. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 5% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |