Refolding Record:
| Protein | |
|---|---|
| Protein Name | Phospholipase A2 |
| Abbreviated Name | PLA2 |
| SCOP Family | Vertebrate phospholipase A2 |
| Structure Notes | |
| Organism | Cobra (naja naja naja) |
| UniProt Accession | P15445 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 120 |
| Molecular Weight | 13345.8 |
| Pi | 4.92556 |
| Molecular Weight | 13345.8 |
| Disulphides | 7 |
| Full Sequence |
NLYQFKNMIKCTVPSRSWWDFADYGCYCGRGGSGTPVDDLDRCCQVHDNCYNEAEKISGC
WPYFKTYSYECSQGTLTCKGDNNACAASVCDCDRLAAICFAGAPYNDNNYNIDLKARCQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kelley MJ, Crowl RM, Dennis EA (1992) Biochim Biophys Acta., 1118, 107-115 |
| Project Aim | Structure-Function |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | MC1061 |
| Expression Temp | 42.0 |
| Expression Time | 5h |
| Expression Vector | pRC25 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Freeze-thaw |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM Tris-HCl, 10mM DTT, 5mM EDTA, 2% Triton X-100, 2M urea, pH 8.5 |
| Solubilization Buffer | 50mM Tris-HCl, 10mM DTT, 5mM EDTA, 8M guanidinium chloride, pH 8.5 |
| Refolding Buffer | 50mM Tris-HCl, 1mM EDTA, 0.1% Triton X-100, 5mM cysteine, 10mM CaCl2 |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 0.01 mg/ml |
| Refolding Time | 260h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The washed inclusion bodies were solubilized and the target protein further purified by size exclusion. The fractions containing the target protein were diluted to 1M guanidine with 50mM Tris-HCl, 1mM EDTA, 0.1% Triton X-100 (pH 8.5) and subsequently dialysed against 100 vols of the same buffer for 60h at 4C. At this time, the protein was diluted to 0.01 mg/ml and 5mM cysteine and 10mM CaCl2 were added and the solution was left to incubateat room temperature. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 0.8mg/L culture |
| Purity | |
| Notes | |