Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lignin peroxidase |
| Abbreviated Name | Lip |
| SCOP Family | CCP-like |
| Structure Notes | |
| Organism | Phanerochaete chrysosporium |
| UniProt Accession | Q01787 |
| 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 | 377 |
| Molecular Weight | 39130.8 |
| Pi | 4.30605 |
| Molecular Weight | 39130.8 |
| Disulphides | 4 |
| Full Sequence |
MALKQLAAAVALALSIQAAQGAAVKEKRATCSNGATVGDASSCAWFDVLDDIQQNLFNGA
QCGAEAHESIRLVFHDAIAISPALESQGKFGGGGADGSIILFDDIETNFHPNIGLDEIVN
LQKPFIQKHGVTPGDFIAFAGAVAMSNCPGAPQMNFFTGRAPATQAAPDGLVPEPFHTVD
QIISRVNDAGEFDELELVWMLSAHSVAAANDVDPTIQGLAFDSTPGVFDSQFFVETQLRG
TAFPGSGGNQGEVESPLPGEMRLQSDSSIARDSRTACEWQSFVNNQSKLVSDFQFIFLAL
TQLGENPDAMTDCSDVIPISKPVPNNVPFSFFPAGKTMADVEQACAETPFPTLTTLPGPE
TSVQRIPPPGA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Doyle WA, Smith AT. (1996) Biochem J., 315, 15-19 |
| Project Aim | Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | DH5 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pFLAG1 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 20mM Tris-HCl, 1mM EDTA, 2mM DTT, 1% Triton X-100, pH 8.0 |
| Solubilization Buffer | 50mM Tris-HCl, 1mM EDTA, 2mM DTT, 6M urea pH 8.0 |
| Refolding Buffer | 20mM Tris-HCl, 0.05mM EDTA, 0.1mM DTT, 5mM CaCl2, 10 micromolar bovine haemin, 2.1M urea, 0.7mM GSSG |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.3 |
| Refolding Temperature | 20.0 |
| Protein Concentration | 0.1 mg/ml |
| Refolding Time | 16h |
| Redox Agent | GSSG/DTT |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Some purification of the LipP* pellets was achieved by washing with 20 mM Tris/HCl (pH 8.0)/1 mM EDTA/2 mM DTT/1% (v/v) Triton X-100 and re-centrifugation at 25000 gmax. for 30 min. Samples to be used for small-scale test folds were washed once, whereas those for large-scale enzyme preparation were washed on average three times. Pellets were then resuspended in 50 mM Tris/HCl (pH 8.0)/1 mM EDTA/2 mM DTT/6 M urea. Prior to large-scale folding the resuspended enzyme was adjusted to 30 mM DTT and incubated at 37 °C for 1 h, before being gel-filtered (Sephadex G-25; PD10 column; Pharmacia) back into the starting buffer. Conditions initially used for larger-scale preparative folds were identified from the small-scale experiments above. These were: 2.1 M urea, 0.7 mM GSSG, 100 mg/ml LipP* protein at pH 8.3. Folding took place over 16 h at 20?5 °C, after which the mixture was concentrated to approximately one-sixth of its initial volume in an Amicon concentrator. A 16 h dialysis against 20 mM sodium acetate, pH 4.3, containing 1 mM CaCl2, was followed by centrifugation at 25000 gmax. for 30 min to remove insoluble material. The activated enzyme was then dialysed against 10 mM sodium succinate, pH 6.0, containing 1 mM CaCl2. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 1% |
| Purity | 80% |
| Notes | |