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 | Ambert-Balay, K., Fuchs, S. M., and Tien, M. (1998) Biochemical and Biophysical Research Com, 251, 283-286 |
| 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 | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 5h |
| Expression Vector | pET21a |
| 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 | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 50 mM TrisHCl pH 8.02 mM EDTA, 2 mM DTT and 6 M urea |
| Refolding Buffer | 50 mM Tris-HCl pH 8.0 and 100 mM CaCl2 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 12 |
| Redox Agent | GSSG |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Cells were induced by the addition of 0.4 mM IPTG and incubated for 1 h. After which rifiampicin was added to a final concentration of 20 mg/ml. Cells were then incubated for a further 4 hours. After incubation the cells were centrifuged and resuspended in 1/100 of the original culture volume with a solution containing 2 mM EDTA, 10 mM DTT and 0.1 mM PMSF in 50 mM TrisHCl, pH 8. Lysozyme and Triton X-100 equivalent to 100 microg/ml and 1% respectively. Cells were then incubated for a further 30 minutes at 30 degC. Cells were then lysed by sonication. (5 cycles for 30 seconds at 3.5 power and 50 % pulse.) Isolated inclusion bodies were then placed in solubilisation buffer. After an incubation of 4-6 hours 0.7 mM GSSG was added before two overnight dialyses in refolding buffer. The forming precipitate was then removed by centrifugation (12 500g, at 4 degC for 15 min). |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 150 microg/ml |
| Purity | |
| Notes | Lignin Peroxidase H8 precurser MASMTGGQQMGAGSE AAAVIEKRAT CSNGKTVGDA SCCAWFDVLD 50 DIQQNLFHGG QCGAEAHESI RLVFHDSIAI SPAMEAQGKF GGGGADGSIM 100 IFDDIETAFH PNIGLDEIVK LQKPFVQKHG VTPGDFIAFA GRVALSNCPG 150 APQMNFFTGR APATQPAPDG LVPEPFHTVD QIINRVNDAG EFDELELVWM 200 LSAHSVAAVN DVDPTVQGLP FDSTPGIFDS QFFVETQLRG TAFPGSGGNQ 250 GEVESPLPGE IRIQSDHTIA RDSRTACEWQ SFVNNQSKLV DDFQFIFLAL 300 TQLGQDPNAM TDCSDVIPQS KPIPGNLPFS FFPAGKTIKD VEQACAETPF 350 PTLTTLPGPE TSVQRIPPPP GA Number of amino acids: 367 Molecular weight: 39034.7 Theoretical pI: 4.44 |