Refolding Record:
| Protein | |
|---|---|
| Protein Name | Chitin deacetylase |
| Abbreviated Name | Chitin deacetylase |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Colletotrichum lindemuthianum |
| UniProt Accession | Q6DWK3 |
| 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 | 254 |
| Molecular Weight | 27808.0 |
| Pi | 5.91848 |
| Molecular Weight | 27808.0 |
| Disulphides | 0 |
| Full Sequence |
MDFSTLLGAA ATAALAGSTN ASPLARRQVP VGTPILQCTQ PGLVALTYDD GPFTFTPQLL DILKQNDVRA TFFVNGNNWA NIEAGSNPDT IRRMRADGHL VGSHTYAHPD LNTLSSADRI SQMRHVEEAT RRIDGFAPKY MRAPYLSCDA GCQGDLGGLG YHIIDTNLDT KDYENNKPET THLSAEKFNN ELSADVGANS
YIVLSHDVHE QTVVSLTQKL IDTLKSKGYR AVTVGECLGD APENWYKA HHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Tokuyasu K, Ohnishi-Kameyama M, Hayashi K, Mori Y (1999) J Biosci Bioeng, 87, 418-423 |
| Project Aim | Folding |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | M15(pREP4) |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pQE60 |
| Expression Protocol | Cells were grown at 37degC in the dark until OD600 reached 0.6. 1mM IPTG was added and cells were incubated a further 4h. The culture was centrifuged (6500g, 15min, 4degC) and the pellet was resuspended in 4ml of 20mM sodium tetraborate/HCl pH 8.5. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 20mM sodium tetraborate/HCl pH 8.5, 8M urea |
| Refolding Buffer | 20mM sodium tetraborate/HCl pH 8.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cells were sonicated for 4min and the lysate centrifuged (9000g, 15min, 4degC). The pelleted inclusion bodies were solubilized in 1ml solubilization buffer. The protein was then diltued into refolding buffer, followed by purification by nickel-affinity chromatography |
| Refolding Assay | Amino acid sequencing |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |