Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lipase chaperone |
| Abbreviated Name | LipB |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Burkholderia cepacia (Pseudomonas cepacia) |
| UniProt Accession | Q6B4I0 |
| 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 | 344 |
| Molecular Weight | 36321.7 |
| Pi | 9.03 |
| Molecular Weight | 36321.7 |
| Disulphides | Unknown |
| Full Sequence |
MTARGGRAPLARRAVVYGAVGLAAVAGVAMWSGAGRHGGTGASGEPPDASAARGPAAAPPQAAVPASTSLPPSLAGSSAPRLPLDAGGHLAKSRAVRDFFDYCLTAQSDLSAAGLDAFVVREIAAQLDGTVAQAEALDVWHRYRAYLDALAKLRDAGAVDKSDLGALQLALDQRASIAYRTLGDWSQPFFGAEQWRQRYDLARLKIAQDPALTDAQKAERLAALEQQMPADERAAQQRVDRQRAAIDQIAQLQKSGATPDAMRAQLTQTLGPEAAVRVAQMQQDDASWQSRYADYAAQRAQIESAGLSPQDRDAQIAALRQRMFTKPGEAVRAASLDRGAGSAR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Jung S, Park S. (2008) Biotechnol Lett, 1, 1 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | E coli |
| Expression Strain | n/a |
| Expression Temp | 25.0 |
| Expression Time | 6 h |
| Expression Vector | pBADgIIIa |
| Expression Protocol | One ml of E. coli overnight culture was added to 100 ml LB medium, and incubated at 37°C and 200 rpm to an OD600 of 0.5. Protein expression was induced by addition of 1 ml arabinose (2% w/v) and the expression culture was incubated for 6 h at 25°C and 200 rpm. When the OD600 reached ~1.5, the cells were harvested by centrifugation (15 min, 3,800g, 4°C) and the supernatant was discarded. The cell pellet (~0.8 g) was resuspended in 4 ml of the lysis buffer (NaH2PO4, 50 mM; NaCl, 300 mM; imidazole, 10 mM; pH 8.0 adjusted with NaOH) and then sonication (4 kHz, five times of 20 s pulse with 30 s interval) was applied. After centrifugation (10 min, 10,000g, 4°C), the cell debris and the supernatant was separated for further experiments. For the expression of B. cepacia lipase, the cell debris was dissolved in 2 ml 8 M urea and the concentration (2.6 mg/ml) and purity of the resulting protein were assessed by the Bradford assay and SDS-PAGE, respectively. This solution of B. cepacia lipase was used for refolding experiments without further purification. |
| Method of Induction | Arabinose |
| Cell Density at Induction | OD 1.5 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Chaperone-assisted refolding |
| Wash Buffer | NaH2PO4, 50 mM; NaCl, 300 mM; imidazole, 10 mM; pH 8.0 adjusted with NaOH |
| Solubilization Buffer | 8 M urea |
| Refolding Buffer | 2 mM CaCl2 in 5 mM BES, pH 7.2 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.2 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | One ml lipase-containing urea solution (total protein content: 2.6 mg/ml) obtained from solubilization of the inclusion bodies was added to 50 ml of the refolding buffer (2 mM CaCl2 in 5 mM BES, pH 7.2) in the presence of 500 mg of the chaperone-conjugated magnetic beads, and the mixture was incubated for 24 h at 4°C. A control without magnetic beads was also performed using 0.43 ml of the soluble chaperone (4.3 mg/ml). After removal of the magnetic beads, the supernatant was concentrated to 1 ml and lipase activity in a 50 μl aliquot was measured by monitoring the hydrolysis of p-nitrophenylacetate as previously described (Jung and Park 2008). The chaperone-conjugated magnetic beads collected by centrifugation or magnetism were recycled by washing three times with 15 ml of BES buffer (5 mM, pH 7.2). |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |