Refolding Record:
| Protein | |
|---|---|
| Protein Name | Subtilisin BPN |
| Abbreviated Name | Subtilisin BPN |
| SCOP Family | Subtilases |
| Structure Notes | |
| Organism | Bacillus amyloliquefaciens |
| UniProt Accession | P00782 |
| 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 | 388 |
| Molecular Weight | 39181.2 |
| Pi | 9.13169 |
| Molecular Weight | 39181.2 |
| Disulphides | 0 |
| Full Sequence |
MRGKKVWISLLFALALIFTMAFGSTSSAQAAGKSNGEKKYIVGFKQTMSTMSAAKKKDVI
SEKGGKVQKQFKYVDAASATLNEKAVKELKKDPSVAYVEEDHVAHAYAQSVPYGVSQIKA
PALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETNPFQDNNSHGTHVAGTVA
ALNNSIGVLGVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSA
ALKAAVDKAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVG
PELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWTNTQVRSSLENT
TTKLGDSFYYGKGLINVQAAAQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Almog, O., Gallagher, T., Tordova, M., Hoskins, J., Bryan, P., and Gilliland, G. (1998) Proteins: Structure, Function, and Genetics, 31, 21-32 |
| Project Aim | Structural Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3 |
| Expression Vector | pG5 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 0.02 M HEPES, pH 7 |
| Solubilization Buffer | 0.02 M HEPES pH 7, 2 M urea |
| Refolding Buffer | 0.1 M KPi, pH 7 |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 12h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | 1 L of LB medium containing 100mg/ml ampicillin was inoculated with cells and grown at 37 degC, 250 rpm in 2-1 baffled flasks to an OD600 of 1.0. Cells were then induced and allowed to grow for a further 3 hours at 37 degC and 250rpm to an OD600 of 1.8. Cells were recovered by centrifugation and stored at -70degC. 9.7g of thawed cells were resuspended in 50ml of 0.1M NaCl, 0.001M EDTA, 0.001M PMSF and 0.05 M Tris pH 8.. The cells were then lysed by French press and 125ml of DNase I (10 mg/ml) was added and allowed to incubate for 15 min. Inclusion bodies were recovered via centrifugation at 10 000f, 4 degC for 20min. Pellet was washed thrice with 60 ml washing buffer and resuspended in 50 ml solubilisation buffer before being frozen on dry ice. After thawing at room temperature the solution was centrifuged at 10 000g, 4 degC for 10 min and supernatant applied to Productive DE cartridge, previously equilibrated with 2 M Urea, 0.02 M HEPES pH 7. Refolding was preformed using a dialysis method against 3.5 L of refolding buffer. Refolded Stb70 was isolated using chromatography using an affinity agarose column. 4.5 mL ALAL-agarose column was equilbrated with 0.02 M HEPES, pH 7. The column was washed with 20 ml of 1 M NaCl, 0.02 M HEPES, pH 7 followed by 10 ml of 1 ml of 0.02 M HEPES pH 7. Sbt70 was eluted with 0.05 M triethylamine, pH 11.1. |
| Refolding Assay | Crystallography |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVAGGASFVPSETNPFQD NNSHGTHVAGTVLAVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGP SGSAALKAAVDKAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASF SSVGPELDVMAPGVSIVSTLPGNKYGAKSGTAMASPHVAGAAALILSKHPNWTNTQVRSS LENTTTKLGDSFYYGKGLINVEAAAQ Number of amino acids: 266 Molecular weight: 26603.6 Theoretical pI: 6.04 |