Refolding Record:
| Protein | |
|---|---|
| Protein Name | Alkaline protease |
| Abbreviated Name | Alkaline protease |
| SCOP Family | Subtilases |
| Structure Notes | |
| Organism | Aspergillus fumigatus |
| UniProt Accession | O74264 |
| 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 | 296 |
| Molecular Weight | 30505.6 |
| Pi | 7.2281 |
| Molecular Weight | 30505.6 |
| Disulphides | 0 |
| Full Sequence |
MRGSALTTQR GAPWGLGSIS HKGQASTDYI YDTSAGAGTY AYVVDTGINV NHVEFEGRAS LAYNAAGGSH VDSVGHGTHV AGTIGGKTYG VAKKTNLLSV KVFQGESSST SIILDGFNWA ANDIVSKGRT RKAAINMSLG GGYSYAFNNA VENAFDEGVL SVVAAGNENT DASETSPASA PNALTVAAIN RSNARASFSN YGSVVDIFAP GQDILSAWIG SNTATNTISG TSMATPHIVG LSVYLMGLEN LSGPAAVTSR IKQLATNGVV TDTRGSPNKL AYNGNATSRS HHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Moser M, Menz G, Blaser K, Crameri R (1994) Infection and Immunity, 62, 936-942 |
| Project Aim | Structure-Function |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | M15 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pAlp |
| Expression Protocol | Expression of protein was induced during the mid-logarithmic phase of cell growth with 2mM IPTG and cells were incubated a further 4h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Chemical |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M GdnHCl pH 8.0 |
| Refolding Buffer | H2O |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were pelleted and lysed overnight in 100ml solubilization buffer. The cleared lysate was applied to a 12ml Ni2+ chelating affinity resin at room temperature. The protein was eluted with in 8M urea with a pH step gradient from 8.0 to 6.0. The protein eluted at pH 5.5. The protein was renatured by a 1:100 dilution with H2O, resulting in a urea concentration of 80mM. The protein was concentrated by ultrafiltration. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |