Refolding Record:
| Protein | |
|---|---|
| Protein Name | SIK W1 lipase |
| Abbreviated Name | SIK W1 lipase |
| SCOP Family | Bacterial lipase |
| Structure Notes | |
| Organism | Pseudomonas fluorescens |
| UniProt Accession | Q647I6 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 476 |
| Molecular Weight | 49888.2 |
| Pi | 4.77 |
| Molecular Weight | 49888.2 |
| Disulphides | 0 |
| Full Sequence |
MGVFDYKNLGTEASKTLFADATAITLYTYHNLDNGFAVGYQQHGLGLGLPATLVGALLGSTDSQGVIPGIPWNPDSEKAALDAVHAAGWTPISASALGYGGKVDARGTFFGEKAGYTTAQAEVLGKYDDAGKLLEIGIGFRGTSGPRESLITDSIGDLVSDLLAALGPKDYAKNYAGEAFGGLLKTVADYAGAHGLSGKDVLVSGHSLGGLAVNSMADLSTSKWAGFYKDANYLAYASPTQSAGDKVLNIGYENDPVFRALDGSTFNLSSLGVHDKAHESTTDNIVSFNDHYASTLWNVLPFSIANLSTWVSHLPSAYGDGMTRVLESGFYEQMTRDSTIIVANLSDPARANTWVQDLNRNAEPHTGNTFIIGSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFSGHFGQDRIIGYQPTDRLVFQGADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGLGGLWSEGVLIS
|
| Notes | Pseudomonas fluorescens SIK W1 lipase gene ligated into pTTY2 through unknown means. As such sequence refers to sequence of SIK W1 lipase gene |
| Expression | |
|---|---|
| Report | Ahn, J.H., Lee, Y.P., Rhee, J. S. (1997) J Biotechnology, 54, 151-160 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pTTY2 |
| Expression Protocol | Cells were grown in a bioreactor prior to induction. The cells were then harvested and resuspended i |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M urea, 0.1 M DTT, 50 mM TrisHCl, pH 7.5 |
| Refolding Buffer | 5 mM DTT, 10 mM CaCl2, 0.65 M GdnHCl |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no |
| Refolding pH | 6.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 12h |
| Redox Agent | DTT |
| Redox Agent Concentration | 5 mM |
| Refolding Protocol | Solubilizing buffer was added to inclusion bodies and insoluble components removed by centrifugation(12 000rpm, 10 min) Soluble sample underwent Q Spharose fast flow column separation, (column equilibrated with solubilizing buffer) Lipase was eluted using buffer and 0-3M GdnHCl linear gradient. Collect lipase was then dialyzes in 3M GdnHCl and stored at -70degC. Refolding was carried out by dilution with refolding buffer. Succinate buffer and Tris/malate buffer were included at a concentration of 50 mM with pH adjusted to 25degC. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |