Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lysozyme |
| Abbreviated Name | Lysozyme |
| SCOP Family | C-type Lysozyme |
| Structure Notes | |
| Organism | Dog (Canis familiaris) |
| UniProt Accession | P81708 |
| 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 | 14602.6 |
| Pi | 8.6191 |
| Molecular Weight | 14602.6 |
| Disulphides | 4 |
| Full Sequence |
MKIFSKCELAR KLKSMGMDGF HGYSLANWVC MAEYESNFNT QAFNGRNSNG SSDYGIFQLN SKWWCKSNSH SSANACNIMC SKFLDDNIDD DIACAKRVVK DPNGMSAWVA WVKHCKGKDL SKYLASCNL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Koshiba T, Hayashi T, Miwako I, Kumagai I, Ikura T, Kawano K, Nitta K, Kuwajima K (1999) Protein Engineering, 12, 429-435 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pSCREEN 1-b(+) |
| Expression Protocol | no details provided |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 50mM Tris-HCl buffer (pH 8.0) containing 6 M guanidine hydrochloride, 1 mM EDTA and 650 mM 2-mercaptoethanol. |
| Refolding Buffer | 22.7 micromolar reduced thioredoxin, 22.7 micromolar oxygenated thioredoxin, 90 micromolar CaCl2 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 2.3 micromolar |
| Refolding Time | 100h |
| Redox Agent | Thioredoxin |
| Redox Agent Concentration | 50mM |
| Refolding Protocol | The refolding of the wild-type and its mutants purified with Sephacryl S-200 were performed in the presence of thioredoxin (Pigiet and Schuster, 1986). Thioredoxin (10 ml; 50 mM) was preincubated with 5 ml DTT (5 mM) for 30 min and added to 10 ml of oxygenated thioredoxin (50 mM) immediately prior to addition of 2 ml of each lysozyme (25 mM). Incubation was carried out at 25degC for 4 days. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |