Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lysozyme |
| Abbreviated Name | Lysozyme |
| SCOP Family | C-type Lysozyme |
| Structure Notes | |
| Organism | Litopenaeus vannamei |
| UniProt Accession | P81708 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 131 |
| Molecular Weight | 14471.4 |
| Pi | 8.62765 |
| Molecular Weight | 14471.4 |
| Disulphides | 4 |
| Full Sequence |
KIFSKCELARKLKSMGMDGFHGYSLANWVCMAEYESNFNTQAFNGRNSNGSSDYGIFQLN
SKWWCKSNSHSSANACNIMCSKFLDDNIDDDIACAKRVVKDPNGMSAWVAWVKHCKGKDL
SKYLASCNL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | de-la-Re-Vega, E., García-Orozco, K.D., Calderón-Arredondo, S.A., Romo-Figueroa, M.G., Islas-Osuna, M.A., Yepiz-Plascencia, G.M. and Sotelo-Mundo, R.R. (2004) Electronic Journal of Biotechnology, 7, 298-304 |
| Project Aim | Biophysical Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | overnight |
| Expression Vector | pET5a |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | none |
| Solubilization Buffer | 50 mM Tris-HCl pH 7.0, 5 mM EDTA, 8 M Gnd-HCl, 5 mM DTT |
| Refolding Buffer | 55 mM Tris, pH 8.5, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, 264 mM NaCl, 11 mM KCl, 550 mM Gdn-HCl and 1 mM EDTA |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | overnight |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Washed inclusion bodies pellet were dissolved in an extraction buffer (50 mM Tris-HCl pH 7.0, 5 mM EDTA, 8 M Gnd-HCl, 5 mM DTT). Extracted inclusion bodies were centrifuged at 22,000 x g for 2 hrs at 4ºC, and diluted with 50 mM Tris-HCl, pH 7.0, 5 mM EDTA, 5 mM DTT to a final concentration of 4 M Gdn-HCl. The solution was centrifuged again at 22,000 x g for 30 min at 4ºC. Refolding buffer (55 mM Tris, pH 8.5, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, 264 mM NaCl, 11 mM KCl, 550 mM Gdn-HCl and 1 mM EDTA) was used for large scale refolding of denatured Lyz. The refolding was done using 6 kDa cut-off dialysis tubing. Two buffer exchanges of 500 ml each were done at 4ºC, and supernatant was obtained by centrifugation at 20,000 x g for 30 min. This solution contains Lyz, and pure protein was isolated by ultrafiltration through a 30 kDa MW cut-off membrane (Millipore), and then concentrated using a 10 kDa cut-off membrane (Millipore). To ensure removal of contaminants, the sample was dialyzed extensively against phosphate buffer 50 mM, pH 8.0 and Lyz activity was detected. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | |