Refolding Record:
| Protein | |
|---|---|
| Protein Name | Hen egg- white lysozyme |
| Abbreviated Name | HEWL |
| SCOP Family | C-type Lysozyme |
| Structure Notes | |
| Organism | Chicken (Gallus gallus) |
| UniProt Accession | P00698 |
| 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 | 129 |
| Molecular Weight | 14313.1 |
| Pi | 9.32 |
| Molecular Weight | 14313.1 |
| Disulphides | 4 |
| Full Sequence |
KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lu D, Liu Z. (2008) J Phys Chem B, 1, 1 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein expressed and purified in native conformation prior to denaturation and refolding. |
| Expression Host | |
| Expression Strain | |
| 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 | n/a |
| Solubilization Buffer | Tris-HCl buffer (100 mM, pH 8.5) containing 8 M urea, 30 mM DTT, and 1 mM EDTA |
| Refolding Buffer | 100 mM Tris-HCl, pH 8.5, 1 mM EDTA, 1.5 M urea, 0.3 mM GSSG, and 3.0 mM GSH and second buffer 100 mM Tris-HCl, pH 8.5, 1 mM EDTA, 1.5 M urea, 5.7 mM GSSG, and 3.0 mM GSH |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 22.0 |
| Protein Concentration | n/a |
| Refolding Time | 4 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 3 mM -0.3 mM & 3/5.7 mM |
| Refolding Protocol | Protein Refolding at Dynamic Redox Environment. Two-step quick dilution method was performed. In the first step, 40 μL of 7.5 mg/mL denatured-reduced lysozyme was quick diluted by 1.5 mL of refolding buffer containing 100 mM Tris-HCl, pH 8.5, 1 mM EDTA, 1.5 M urea, 0.3 mM GSSG, and 3.0 mM GSH. After 30 min, in the second step the protein solution was further diluted by 1.5 mL of refolding buffer containing 100 mM Tris-HCl, pH 8.5, 1 mM EDTA, 1.5 M urea, 5.7 mM GSSG, and 3.0 mM GSH. Thus the final concentrations of GSSG and GSH are both 3.0 mM. For the control experiments, two-step quick dilution method was also performed with the same refolding buffer containing constant GSSG and GSH. The final lysozyme activity was determined after incubation for 4.0 h. |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | Two-step quick dilution method was performed for refolding so Two refolding buffer used |