Refolding Record:
| Protein | |
|---|---|
| Protein Name | Keratinase |
| Abbreviated Name | Keratinase |
| SCOP Family | Subtilases |
| Structure Notes | |
| Organism | Bacillus licheniformis |
| UniProt Accession | Q53521 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | Keratinase-streptavidin construct |
| Variants | n/a |
| Chain Length | 509 |
| Molecular Weight | 52106.8 |
| Pi | 7.3 |
| Molecular Weight | 52106.8 |
| Disulphides | 0 |
| Full Sequence |
AQPAKNVEKDY IVGFKSGVKT ASVKKDIIKE SGGKVDKQFR IINAAKAKLD KEALKEVKND PDVAYVEEDH VAHALAQTVP YGIPLIKADK VQAQGFKGAN VKVAVLDTGI QASHPDLNVV GGASFVAGEA YNTDGNGHGT HVAGTVAALD NTTGVLGVAP SVSLYAVKVL NSSGSGSYSG IVSGIEWATT NGMDVINMSL GGASGSTAMK QAVDNAYARG VVVVAAAGNS GSSGNTNTIG YPAKYDSVIA VGAVDSNSNR ASFSSVGAEL EVMAPGAGVY STYPTNTYAT LNGTSMVSPH VAGAAALILS KHPNLSASQV RNRLSSTATY LGSSFYYGKG LINVEAAAQ DPSKDS KAQVSAAEAG ITGTWYNQLG STFIVTAGAD GALTGTYESA VGNAESRYVL TGRYDSAPAT DGSGTALGWT VAWKNNYRNA HSATTWSGQY VGGAEARINT QWLLTSGTTE ANAWKSTLVG HDTFTKVKPS AASIDAAKKA GVNNGNPLDA VQQ
|
| Notes | approximated sequence only. streptavidin sequence based on UniProt SAV_STRAV A range of other constructs with or without the Pre and Pro regions of keratinase also produced |
| Expression | |
|---|---|
| Report | Wang JJ, Swaisgood HE, Shih JC. (2003) Biotechnology and Bioengineering, 81, 421-9 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 37.0 |
| Expression Time | 2-4h |
| Expression Vector | pBSTP |
| Expression Protocol | Expression was induced by the addition of 0.1mM IPTG and cells were incubated a further 2-4h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Osmotic shock + sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 6N GdnHCl, 50mM TrisHCl pH 8.0 |
| Refolding Buffer | 0.5M (NH4)2SO4, 10mM TrisHCl, 10mM Ha2HPO4, 1mM CaCl2 pH 7.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.1 mg/ml |
| Refolding Time | overnight |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were treated by osmotic shock in 20% sucrose, then sonicated. The cell lysate was centrifuged (8000g, 4degC, 10min) and the inclusion bodies dissolved in solubilization buffer. The protein was refolded by dialysis at 4degC overnight. |
| Refolding Assay | Enzyme activity,Unspecified,Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | Various refolding buffer conditions trialled, see paper for more details. |