Refolding Record:
| Protein | |
|---|---|
| Protein Name | Hen egg- white lysozyme |
| Abbreviated Name | HEWL |
| SCOP Family | C-type Lysozyme |
| Structure Notes | |
| Organism | Hen |
| UniProt Accession | P00698 |
| 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 | 130 |
| Molecular Weight | 14313.1 |
| Pi | 9.32 |
| Molecular Weight | 14313.1 |
| Disulphides | 4 |
| Full Sequence |
KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDG
NGMNAWVAWRNRCKGTDVQAWIRGCRL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Rozema D, Gellman SH (1996) Biochemistry, 35, 15760-15771 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein expressed and purified in native conformation prior to denaturation and refolding. |
| Expression Host | None |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 0 |
| Expression Vector | |
| Expression Protocol | |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 8.7 M GdmCl, 143 mM Tris sulfate, and 43 mM DTT |
| Solubilization Buffer | 0.071 mg/mL lysozyme, 8.6 mM GdmCl, 0.04 mM DTT, 0.071 mM CuSO4, 1.43 mM EDTA, and 0 or 5.7 mM CTAB |
| Refolding Buffer | 0.05 mg/mL lysozyme, 6.0 mM GdmCl, 0.03 mM DTT, 0.05 mM CuSO4, 1.0 mM EDTA, 0 or 4.0 mM CTAB, and 4.8 mM -cyclodextrin |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 40 |
| Redox Agent | DTT |
| Redox Agent Concentration | 0.03 mM |
| Refolding Protocol | Copper sulfate-catalyzed oxidation of denatured-reduced lysozyme was performed by dilution of 50 mg/mL of denatured-reduced lysozyme in 6 M GdmCl and 30 mM DTT to 0.071 mg/mL lysozyme, 8.6 mM GdmCl, 0.04 mM DTT, 0.071 mM CuSO4, 1.43 mM EDTA, and 0 or 5.7 mM CTAB. The solutions were allowed to sit for various periods of time, and then -cyclodextrin was added to make a solution of 0.05 mg/mL lysozyme, 6.0 mM GdmCl, 0.03 mM DTT, 0.05 mM CuSO4, 1.0 mM EDTA, 0 or 4.0 mM CTAB, and 4.8 mM B-cyclodextrin. After sitting 40 h, the solutions were assayed for enzymatic activity. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | β-cyclodextrin |
| Additives Concentration | 4.8 mM |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | Please note temperature is not specified assumed it is in room temperature |