Refolding Record:
| Protein | |
|---|---|
| Protein Name | Chlorate reductase subunit gamma |
| Abbreviated Name | ClrC |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Ideonella dechloratans |
| UniProt Accession | P60000 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 212 |
| Molecular Weight | 22902.1 |
| Pi | 9.96 |
| Molecular Weight | 22902.1 |
| Disulphides | Unknown |
| Full Sequence |
AQGAVPQAQRIIRVLSVAGGDAASPQAAVWKKAPTTQVTLLTAFPGHISIVGTAATQKLAAQAVRASGRLFVRLAWSDRTANTVMKDTDQFLDGAAVEFPVNGKVATLPFMGDPVNVVNVWHWRADGRTLNLLAKGFGTSTPVPTEDLRSASVRTGDGWEVVLSRPLRVKAEEGANLQGRRTMPIGFAAWDGENQERDGLKAVTMEWWQLRF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Karlsson J, Nilsson T. (2005) Protein Expression and Purification, 41, 306-312 |
| Project Aim | Over expression & Renaturation |
| Fusion | GST |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 30.0 |
| Expression Time | 4 h |
| Expression Vector | pJK13 |
| Expression Protocol | A 5 ml overnight culture with BL21/pJK13 was used to inoculate 1 L LB medium containing 100 mg/L ampicillin. The culture was incubated at 30 °C to an optical density (OD600) of 0.5 and then induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG, Fluka). After 4 h, the cells were harvested by a 20 min centrifugation at 5000g. The pellet was stored overnight in −20 °C. Expression and purification was monitored by SDS–PAGE and by immunoblotting with Anti-GST Antibody (Amersham Biosciences). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 25 mM sodium phosphate at pH 7.0, containing 0.5 mM EDTA, 1 mM DTT, and 8 M urea |
| Refolding Buffer | 55 mM Tris, pH 8.2, 21 mM NaCl, 0.88 mM KCl, 0.44 M arginine, 1 mM EDTA, 10 μM PEG, and 10 mM DTT |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.2 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 h |
| Redox Agent | DTT |
| Redox Agent Concentration | 10 mM |
| Refolding Protocol | Refolding of GST/ClrC fusion protein For refolding trials, the Pro-Matrix Protein Kit (Pierce Biotechnology) was used. Screening for optimal refolding conditions was performed according to the instructions. Heme solution was prepared fresh by dissolving 10 mg hemin chloride (MP Biomedicals) in 1 ml dimethyl sulfoxide (DMSO), followed by rapid vortexing. The solution was centrifuged for 10 min (14,000g). The heme concentration was determined spectrophotometrically using the extinction coefficient ε385 = 56 cm−1 mM−1 [21]. All refolding studies were performed at 4 °C. The optimization of the refolding was based on turbidity measurement at 360 nm and by monitoring the GST activity (GST Gene Fusion System Handbook, Amersham Biosciences). The optimized refolding buffer (55 mM Tris, pH 8.2, 21 mM NaCl, 0.88 mM KCl, 0.44 M arginine, 1 mM EDTA, 10 μM PEG, and 10 mM DTT), in the following referred to as buffer R2, was used at 4 °C. Urea-solubilized protein (0.5 ml and 5.4 mg/ml) was added dropwise (approximately 12 drops/min) to 10 ml buffer R2 under rapid vortexing. The diluted fusion protein was kept for 24 h at 4 °C followed by centrifugation at 20,000g for 10 min. Buffer exchange from buffer R2 to phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2PO4, and 2 mM KH2PO4, pH 7.4) was performed on a PD10 column (Amersham Biosciences) followed by centrifugation (10 min, 20,000g). The refolded fusion protein was stored at 4 °C until further characterization. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.44 M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |