Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lipase from Burkholderia cepacia |
| Abbreviated Name | Lipase |
| SCOP Family | Bacterial lipase |
| Structure Notes | |
| Organism | Burkholderia cepacia KWI-56 |
| UniProt Accession | P22088 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 320 |
| Molecular Weight | 33160.1 |
| Pi | 5.56 |
| Molecular Weight | 33160.1 |
| Disulphides | 1 |
| Full Sequence |
AAGYAA TRYPIILVHG LSGTDKYAGV LEYWYGIQED LQQNGATVYV ANLSGFQSDD GPNGRGEQLL AYVKTVLAAT GATKVNLVGH SQGGLSSRYV AAVAPDLVAS VTTIGTPHRG SEFADFVQDV LAYDPTGLSS SVIAAFVNVF GILTSSSHNT NQDALAALQT VTTARAATYN QNYPSAGLGA PGSCQTGAPT ETVGGNTHLL YSWAGTAIQP TLSVFGVTGA TDTSTLPLVD PANVLDLSTL ALFGTGTVMI NRGSGQNDGL VSKCSALYGK VLSTSYKWNH LDEINQLLGV RGAYAEDPVA VIRTHANRLK LAGV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Yang J, Koga Y, Nakano H, Tamane T (2002) Protein Engineering, 15, 147-152 |
| 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 | BL21(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pRSET |
| Expression Protocol | Cells were grown in 800ml LB medium containing 1% glucose, 50microg/ml ampicillin and 25microg/ml chloramphenicol at 37degC until OD600 reached 0.6. IPTG was added to 0.4mM and cells were grown a further 3h. Cells were harvested by centrifugation and then suspended in 50mM TrisHCl, 30mM NaCl |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM TrisHCl pH 8.0, 30mM NaCl |
| Solubilization Buffer | 10mM TrisHCl, 8M urea, 0.1M sodium phosphate, pH 8.0 |
| Refolding Buffer | distilled water with lipase activator |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 20h |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a,n/a |
| Refolding Protocol | Cells were sonicated, and centrifuged (300g, 10min), and the supernatant was collected as the crude cell extract. Then soluble and insoluble proteins were spearated by centrifugation (8000g, 20min). The inclusion bodies collected were resuspended in 1M sucrose and centrifuged (8000g, 20min). The pellets were then washed twice with wash buffer. The purified inclusion bodies were dissolved in solubilization buffer at room temperature for 1h with mild stirring. Following centrifugation (8000g, 20min), the supernatant was refolded by the dilution of 100microL of protein wolution into 100ml of distilled water in the presence of the same amount of lipase activator. The protein was refolded at 4degC for 20h. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |