Refolding Record:
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 3 |
| Molecular Weight | 203.2 |
| Pi | 5.52 |
| Molecular Weight | 203.2 |
| Disulphides | Unknown |
| Full Sequence |
n/a
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | St John RJ, Carpenter JF, Randolph TW. (2002) Biotechnol Prog, 18, 565-571 |
| 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 | High Pressure |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M GdnHCl and 40 mM dithiothreitol (DTT) |
| Refolding Buffer | 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1.0 M GdnHCl, 2 mM DTT, 6 mM GSSG |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 24.0 |
| Protein Concentration | n/a |
| Refolding Time | 50 h |
| Redox Agent | GSSG/DTT |
| Redox Agent Concentration | 6/2 mM |
| Refolding Protocol | To gain insight into where during the pressurization/depressurization cycle lysozyme refolding occurs, we studied the effects of pressure on the conformation of native lysozyme in the refolding buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1.0 M GdnHCl, 2 mM DTT, 6 mM GSSG). Figure 5a displays the second derivative UV spectra. The native lysozyme peaks are red-shifted relative to Tyr in water (31), indicating that the Tyr residues that dominate the absorbance spectrum are in the hydrophobic interior of the protein. As pressure increased, the peaks of the spectrum of lysozyme decreased in intensity and shifted slightly to longer wavelengths |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 80% |
| Purity | n/a |
| Notes | n/a |