Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lysozyme |
| Abbreviated Name | Lysozyme |
| SCOP Family | C-type Lysozyme |
| Structure Notes | |
| Organism | Bacteriophage K11 |
| UniProt Accession | Q3ZFI3 |
| 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 | 151 |
| Molecular Weight | 16710.9 |
| Pi | 8.48 |
| Molecular Weight | 16710.9 |
| Disulphides | Unknown |
| Full Sequence |
MAKVQFTKRQETSQFFVHCSATKANMDVGVREIRQWHKEQGWLDVGYHFIIRRDGTVEAGRDQDAVGSHVKGYNSTSVGVCLVGGIDAKGNPEANFTPQQMSALNGVLHELRGTYPKAVIMAHHDVAPKACPSFDLQRWVKTGELVTSDRG
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Junn HJ, Youn J, Suh KH, Lee SS. (2005) Protein Expression and Purification, 42, 78-84 |
| Project Aim | Cloning and expression |
| Fusion | N-terminal T7 tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | XLBlue |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pETtacKLYS |
| Expression Protocol | A 400-ml culture of XL1-blue/pET-tacKLYS was grown at 37 °C in a shaking flask with LB medium. When the culture reached A600nm = 0.5, the tac promoter was induced with 0.5 mM IPTG. After an additional 4-h growth, cells were harvested by centrifugation and resuspended in 200 ml of the above buffer A. After the cells were sonicated using a multitip with 50% power for ten 30-s intervals on ice, the lysates (crude extract) were centrifuged at 18,000 rpm (Sorvall SS-34 rotor) for 20 min, and the pellets (inclusion body) were collected. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | phosphate-buffered saline (PBS), 15% glucose, 5 mM EDTA, 1% Triton X-100, and 0.5 mM PMSF |
| Solubilization Buffer | 50 mM Tris–HCl, pH 8.0, 5 M guanidine–HCl, and 5 mM EDTA |
| Refolding Buffer | 50 mM Tris–HCl, pH 8.0, 1% Triton X-100, 8 mM reduced glutathione (GSH), 1 mM oxidized glutathione (GSSG), 0.5 mM PMSF, and 0.3 M arginine |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 8/1 mM |
| Refolding Protocol | he pellets were extracted with washing buffer consisting of phosphate-buffered saline (PBS), 15% glucose, 5 mM EDTA, 1% Triton X-100, and 0.5 mM PMSF and recentrifuged at 18,000 rpm (Sorvall SS-34 rotor) for 20 min. After three times washing, the pellets were resuspended in denaturation buffer consisting of 50 mM Tris–HCl, pH 8.0, 5 M guanidine–HCl, and 5 mM EDTA and incubated on ice for 1 h. The protein solubilized in denaturation buffer was slowly diluted with 10 times volume of renaturation buffer consisting of 50 mM Tris–HCl, pH 8.0, 1% Triton X-100, 8 mM reduced glutathione (GSH), 1 mM oxidized glutathione (GSSG), 0.5 mM PMSF, and 0.3 M arginine. After refolding the denatured protein through this process, the soluble fractions were dialyzed against a buffer consisting of 50 mM Tris–HCl, pH 8.0, 0.1% Triton X-100 for overnight and against a storage buffer consisting of 50 mM Tris–HCl, pH 8.0, 1 mM dithiothreitol, 1 mM EDTA, 0.1% Triton X-100, and 50% glycerol. The fractions were identified in each purification step by SDS–PAGE and protein concentration and bacterial cell wall lysis activity were measured. The purified enzyme was stored at −70 °C until used. |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.3 M |
| Refolding Yield | 5% |
| Purity | n/a |
| Notes | n/a |