Refolding Record:
| Protein | |
|---|---|
| Protein Name | Pro-subtilisin |
| Abbreviated Name | n/a |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Bacillus subtilis |
| UniProt Accession | Q6IT79 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 382 |
| Molecular Weight | 39095.1 |
| Pi | 9.23 |
| Molecular Weight | 39095.1 |
| Disulphides | Unknown |
| Full Sequence |
MRGKKVWISLLFALALIFTMAFGSTSPAQAAGKSNGEKKYIVGFKQTMSTMSAAKKKDVISEKGGKVQKQFKYVDAASATLNEKAVKELKKDPSVAYVEEDHVAQAYAQSVPYGVSQIKAPALHSQGFTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETNPFQDNNSHGTHVAGTVAALNNSVGVLGVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGTSGGSSTVGYPGKYPSVIAVGAVNSSNQRASFSSVGSELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDAFYYGKGLINVQAAAQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Li Y, Inouye M. (1996) J Mol Biol, 262, 591-4 |
| Project Aim | Undefined |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | pET16B |
| Expression Protocol | The expression protocol,time and temperature are not stated in this paper |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | guanidine-Hcl |
| Refolding Buffer | 10 mM Tris-HCl (pH 7.0), 0.5 M (NH4 )2 SO4 , 1 mM CaCl2 , 1 mM DTT |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 7.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 1 mM |
| Refolding Protocol | Autoprocessing of His10-prosubtilisin(S221C). The protein was purified in the presence of 5 M urea as described (Li & Inouye, 1994) and then subjected to step-wise renaturation. The renaturing buffer is 10 mM Tris-HCl (pH 7.0), 0.5 M (NH4 )2 SO4 , 1 mM CaCl2 , 1 mM DTT. The protein was dialyzed against this buffer containing decreasing amounts of urea. Samples were taken two hours after 4 M urea (lane 1), 2 M (lane 2), 1 M (lane 3), 0.5 M (lane 4), 0 M (lane 5), and OM overnight (lane 6) dialysis and analyzed on a SDS/polyacrylamide gel. X, Y and Z indicate the position of His10-prosubtilisin(S221C), mature subtilisin(S221C) and His10-propeptide, respectively. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | (NH4)2SO4 |
| Additives Concentration | 0.5 M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |