Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lysozyme |
| Abbreviated Name | Lysozyme |
| 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 | 130 |
| Molecular Weight | 14313.1 |
| Pi | 9.32 |
| Molecular Weight | 14313.1 |
| Disulphides | Unknown |
| Full Sequence |
KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDG
NGMNAWVAWRNRCKGTDVQAWIRGCRL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Dong XY, Huang Y, Sun Y. (2004) J Biotechnology, 114, 135-142 |
| 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 | n/a |
| Solubilization Buffer | 0.1 M Tris–sulfate buffer (pH 8.5) containing 6 M GdmCl and 30 mM DTT |
| Refolding Buffer | 5 mM GSH, 5 mM GSSG, 0.3–1.2 mM DTT, 1 mM EDTA |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 37.0 |
| Protein Concentration | n/a |
| Refolding Time | 3-12 h |
| Redox Agent | GSH/GSSG/DTT |
| Redox Agent Concentration | 5/5/0.3-1.0 mM |
| Refolding Protocol | In the refolding experiments, the denatured-reduced lysozyme solution (20 mg ml−1) was rapidly diluted to 0.1 M Tris–sulfate solution (pH 8.5). The final refolding solution contained 5 mM GSH, 5 mM GSSG, 0.3–1.2 mM DTT, 1 mM EDTA and definite concentrations of lysozyme (0.2–0.8 mg ml−1), GdmCl (0.06–2.2 M) and any of the additives at a final volume of 3 ml. The final concentrations of the redox reagents were in the range of the optimum concentrations suggested by De Bernardez Clark et al. (1998). The renaturation system was incubated at 37 °C in a shaking incubator for a period of 3–12 h (depending on the concentrations of GdmCl and the additives) until the refolding reached equilibrium. During the refolding experiments, 100 μL samples were withdrawn at different time intervals and 10 μL of 0.5 M iodoacetic acid was added immediately to stop additional folding. The quenched sample was used for lysozyme activity assay as described below. The refolding yield was expressed as the percentage of the specific activity of renatured lysozyme relative to that of the native lysozyme. |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |