Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lysozyme |
| Abbreviated Name | Lysozyme |
| SCOP Family | C-type Lysozyme |
| Structure Notes | |
| Organism | Chicken (Gallus gallus) |
| UniProt Accession | Q6LEL2 |
| 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 | 45 |
| Molecular Weight | 4953.0 |
| Pi | 9.69 |
| Molecular Weight | 4953.0 |
| Disulphides | Unknown |
| Full Sequence |
MRSLLILVLCFLPLAALGKVFGRCELAAAMKRHGLDNYRGYSLGN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lanckriet H, Middelberg AP. (2004) Biotechnol Prog, 20, 1861-1866 |
| 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 | not specified |
| Solubility | |
| Refolding | |
|---|---|
| Refolding Method | Dilution/ chaperone-assisted refolding |
| Wash Buffer | n/a |
| Solubilization Buffer | 0.1 M Tris, 1 mM EDTA, 6 M urea and 32 mM DTT, pH 8.1 |
| Refolding Buffer | 0.12 M urea, 0.64 mM DTT, 3 mM cysteine, 0.3 mM cystine, 0.1 M Tris, 1 mM EDTA, and 8.1 or 0 mM CTAB (cetyltrimethylammonium bromide ) at pH 8.1 and 0.12 M urea, 0.64 mM DTT, 3 mM cysteine, 0.3 mM cystine, 0.1 M Tris, 1 mM EDTA, 16.3 or 0 mM B-CD |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.1 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 0.2 mg/ml |
| Refolding Time | 24 h |
| Redox Agent | DTT/cysteine/cystine |
| Redox Agent Concentration | 0.64/3/0.3 mM,0.64/3/0.3 mM |
| Refolding Protocol | Two-Step Batch Dilution Refolding. Standard denatured and reduced lysozyme (50 mg/mL) was diluted 100-fold resulting in a solution containing 0.5 mg/mL lysozyme, 0.12 M urea, 0.64 mM DTT, 3 mM cysteine, 0.3 mM cystine, 0.1 M Tris, 1 mM EDTA, and 8.1 or 0 mM CTAB at pH 8.1. Then, 0.5-mL samples were diluted by adding 0.670 mL of buffer containing 0.12 M urea, 0.64 mM DTT, 3 mM cysteine, 0.3 mM cystine, 0.1 M Tris, 1 mM EDTA, and 16.3 or 0 mM -CD. The final lysozyme concentration in each sample was 0.2 mg/mL. Native lysozyme control samples were never denatured. All samples were left for 24 h before assaying. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | β-cyclodextrin |
| Additives Concentration | 16.30 or 0 mM |
| Refolding Yield | 1%-53% |
| Purity | n/a |
| Notes | In this paper Two step dilution used for refolding Attention the refolding yield without CTAB is 1% and with CTAB + B-CD is 53% |