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 | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | C6S/C127S variant - lacking one disulfide bond |
| Chain Length | 129 |
| Molecular Weight | 14281.0 |
| Pi | 9.54 |
| Molecular Weight | 14281.0 |
| Disulphides | 3 |
| Full Sequence |
KV FGRSELAAAM KRHGLDNYRG YSLGNWVCAA KFESNFNTQA TNRNTDGSTD YGILQINSRW WCNDGRTPGS RNLCNIPCSA LLSSDITASV NCAKKIVSDG NGMNAWVAWR NRCKGTDVQA WIRGSRL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Sawano H, Koumoto Y, Ohta K, Sasaki Y, Segawa S, Tachibana H (1992) FEBS Letters, 303, 11-14 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | AD18 |
| Expression Temp | 37.0 |
| Expression Time | n/a |
| Expression Vector | pYK1 |
| Expression Protocol | Cells were grwon in LB-medium containing 25microg/ml ampicillin and 50microg/ml kanamyciin. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 8M urea 50mM DTT |
| Refolding Buffer | 100mM Tris-acetate, 1mM EDTA, pH 7.8, 6mM GSH, 0.6mM GSSG |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.8 |
| Refolding Temperature | 15.0 |
| Protein Concentration | 3microM |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 6/0.6mM |
| Refolding Protocol | Cells were disrupted by sonication and inclusion bodies were solubilized in solubilization buffer. The protein was purified by cation-exchange and gel filtration chromatography, freeze-dried and stored frozen. The protein was renatured by dilution in refolding buffer, after 2h of refolding, the reaction was terminated by acidification to below pH 5. The protein was then purified further by reverse phase HPLC |
| Refolding Assay | Near-UV Circular Dichroism,Enzyme activity,HPLC |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 20% |
| Refolding Yield | 68% |
| Purity | n/a |
| Notes | Refolding also performed at 37degC and without glycerol, but less successful |