Refolding Record:
| Protein | |
|---|---|
| Protein Name | Triacylglycerol lipase |
| Abbreviated Name | Lipase |
| SCOP Family | Fungal lipases |
| Structure Notes | |
| Organism | Aspergillus niger |
| UniProt Accession | Q0ZAX7 |
| 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 | 270 |
| Molecular Weight | 28892.1 |
| Pi | 4.43 |
| Molecular Weight | 28892.1 |
| Disulphides | Unknown |
| Full Sequence |
SVSTSTLDELQLFAQWSAAAYCSNNIDSDDSNVTCTADACPSVEEASTKMLLEFDLTNNFGGTAGFLAADNTNKRLVVAFRGSSTIKNWIADLGFILQDNDDLCTGCKVHTGFWKAWEAAADNLTSKIKSAMSTYSGYTLYFTGHSLGGALATLGATVLRNDGYSVELYTYGCPRVGNYALAEHITSQGSGANFRVTHLNDIVPRLPPMDFGFSQPSPEYWITSGTGASVTASDIELIEGINSTAGNAGEATVDVLAHLWYFFAISECLL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Shu ZY, Yan YJ, Yang JK, Xu L (2007) biotechnology letters, 29, 1875-1879 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pET28a-lipanl |
| Expression Protocol | The plasmid pET28a-lipanl was transformed into E. coli BL21(De3). The transformant was grown at 37°C in LB liquid medium supplemented with 25 μg kanamycin/ml. After incubation overnight, the cultures were transferred to the new LB liquid media at 1:20 (v/v). The expression of the lipanl was induced by adding IPTG to 2 mM when the cell density (OD600) reached 0.6. The cultivation was continued 4 h and the cells were harvested by centrifugation. The cell pellet was resuspended in 0.05 M Tris/HCl buffer (pH 7.5), and disrupted by ultrasonication in ice-bath for 10 min. The cell lysate was centrifuged at 8,000 g for 10 min at 4°C, and the supernatant and the precipitate of the cell lysate were recovered to be analyzed by SDS-PAGE, respectively. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 600 |
| Cell Disruption Method | ultrasonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ni-NTA agrose chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 0.05 M Tris/HCl buffer (pH 7.5) |
| Solubilization Buffer | not indicated in paper |
| Refolding Buffer | 50 mM Tris/HCl (pH 7.5), 10% (w/v) glycerol, 1% (w/v) glycine, 2 mM l-glutathione (reduced form) and 1 mM l-glutathione (oxidized form) |
| Pre-Refolding Purification | Ni-NTA agrose chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 12 h |
| Redox Agent | GSH |
| Redox Agent Concentration | 2 mM,2 mM,2 mM |
| Refolding Protocol | Purification and refolding of the rANL in vitro The purification of the rANL was carried out by Ni-NTA agrose chromatography according to the instructions, manufacturer’s (Qiagen). DDT was added to the purified rANL solution to 10 mM, and the treatment was continued for overnight at 4°C. The denatured rANL was then diluted to 50 μg/ml with the renaturing buffer composed of 50 mM Tris/HCl (pH 7.5), 10% (w/v) glycerol, 1% (w/v) glycine, 2 mM l-glutathione (reduced form) and 1 mM l-glutathione (oxidized form). The diluted rANL solution was kept sealed for 12 h, and then unsealed for 12 h at 4°C. Following these treatments, the rANL solution was applied to a DEAE-sepharose Fast Flow column (1 × 11 cm) pre-equilibrated with 50 mM Tris/HCl buffer (pH 7.5). After the column was rinsed with 60 ml equilibration buffer, the rANL was eluted with 100 ml 0.3 M NaCl in the same buffer. The fractions corresponding to the eluate peak were collected. Part of the rANL eluate solution was dialyzed against 10 mM CaCl2 in 50 mM Tris/HCl buffer (pH 7.5). |
| Refolding Assay | Protein activity assay |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 10% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |