Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lipase |
| Abbreviated Name | lip4 |
| SCOP Family | Fungal lipases |
| Structure Notes | |
| Organism | Pseudomonas fluorescens |
| UniProt Accession | Q76D31 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 617 |
| Molecular Weight | 64354.1 |
| Pi | 4.58 |
| Molecular Weight | 64354.1 |
| Disulphides | Unknown |
| Full Sequence |
MGVYDYKNFGTADSKALFSDAMAITLYSYHNLDNGFAAGYQHNGFGLGLPATLVTALLGGTDSQGVIPGIPWNPDSEKLALEAVKKAGWTPITASQLGYDGKTDARGTFFGEKAGYSTAQVEILGKYDAQGHLTEIGIAFRGTSGPRENLILDSIGDVINDLLAAFGPKDYAKNYVGEAFGNLLNDVVAFAKANGLSGKDVLVSGHSLGGLAVNSMADLSGGKWGGFFADSNYIAYASPTQSSTDKVLNVGYENDPVFRALDGSNFTGASIGVHDAPKESATDNIVSFNDHYASTAWNLLPFSILNIPTWISHLPTAYGDGMNRVIESKFYDLTSKDSTIIVANLSDPARANTWVQDLNRNAETHKGSTFIIGSDANDLIQGGSGNDYLEGRAGNDTFRDSGGYNIILGGQGSNTLDLQKSVNTFDFANDGAGNLYIRDANGGISITRDIGAIVTKEPGFLWGLFKDDVTHSVTASGLKVGNNLTAYESSVKGSNGADTLKAHAGGDWLFGLDGNDHLIGGAGNDVFVGGAGNDLMESGGGADTFLFNGAFGQDRVVGFTSNDKLVFLGVQGVLPGDDFRAHASAAGQDTVLKFGGDSVTLVGVSLGSLSGDGIVIA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kojima Y, Kobayashi M, Shimizu S. (2003) J Biosci Bioeng, 96, 242-249 |
| Project Aim | Over expression & Renaturation |
| Fusion | N-terminal methionine |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | JM109 |
| Expression Temp | 0.0 |
| Expression Time | 8 h |
| Expression Vector | pLPM101 |
| Expression Protocol | Preparation of inclusion bodies and refolded lipase For the assay of lipase expressed in E. coli JM109, transformants containing each of the recombinant plasmids were grown for 4 h in 100 ml of LB medium supplemented with IPTG to induce the lac promoter. After 8 h of cultivation, the cells were harvested by centrifugation at 5000xg for 10 min at 4°C the resulting pellets were washed once with 50 mM phosphate buffer (pH 7.0) and resuspended in 30 ml of the same ice-cold buffer, and then sonicated for 10 min (19 kHz; Insonator model 201M; Kubota, Tokyo). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM phosphate buffer (pH 7.0) |
| Solubilization Buffer | 50 mM phosphate buffer (pH 7.0) containing 8 M urea and 5% glycerol |
| Refolding Buffer | 50 mM phosphate buffer (pH 7.0), 5% glycerol |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 7.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 13.3 mg |
| Refolding Time | 16 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The pellets were then washed once with the same buffer and dissolved in 8 ml of 50 mM phosphate buffer (pH 7.0) containing 8 M urea and 5% glycerol. After standing for 3 h at 37°C the suspension was diluted to 56 ml with 50 mM phosphate buffer (pH 7.0) containing 5% glycerol and kept for 16 h at 4°C. After this solution was dialyzed against 50 mM phosphate buffer (pH 7.0) containing 5% glycerol, it was concentrated and desalted by ultrafiltration with an Amicon ultrafiltration unit (Millipore, Bedford, MA, USA) and YMlO membranes (exclusion limit, 10 kD cut off) to obtain the refolded lipase. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 5% |
| Refolding Yield | 100% |
| Purity | 1 |
| Notes | n/a |