Refolding Record:
| Protein | |
|---|---|
| Protein Name | Prothermolysin |
| Abbreviated Name | Prothermolysin |
| SCOP Family | Thermolysin-like |
| Structure Notes | |
| Organism | Bacillus thermoproteolyticus |
| UniProt Accession | P00800 |
| 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 | 557 |
| Molecular Weight | 60103.9 |
| Pi | 5.52734 |
| Molecular Weight | 60103.9 |
| Disulphides | 0 |
| Full Sequence |
MKMKMKLASFGLAAGLAAQVFLPYNALASTEHVTWNQQFQTPQFISGDLLKVNGTSPEEL
VYQYVEKNENKFKFHENAKDTLQLKEKKNDNLGFTFMRFQQTYKGIPVFGAVVTSHVKDG
TLTALSGTLIPNLDTKGSLKSGKKLSEKQARDIAEKDLVANVTKEVPEYEQGKDTEFVVY
VNGDEASLAYVVNLNFLTPEPGNWLYIIDAVDGKILNKFNQLDAAKPGDVKSITGTSTVG
VGRGVLGDQKNINTTYSTYYYLQDNTRGNGIFTYDAKYRTTLPGSLWADADNQFFASYDA
PAVDAHYYAGVTYDYYKNVHNRLSYDGNNAAIRSSVHYSQGYNNAFWNGSQMVYGDGDGQ
TFIPLSGGIDVVAHELTHAVTDYTAGLIYQNESGAINEAISDIFGTLVEFYANKNPDWEI
GEDVYTPGISGDSLRSMSDPAKYGDPDHYSKRYTGTQDNGGVHINSGIINKAAYLISQGG
THYGVSVVGIGRDKLGKIFYRALTQYLTPTSNFSQLRAAAVQSATDLYGSTSQEVASVKQ
AFDAVGVK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Marie-Claire C, Roques BP, Beaumont A. (1998) J Biol Chem, 273, 5697-5701 |
| Project Aim | Functional Studies |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | unknown |
| Expression Vector | pRSET C |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Matix assisted dialysis |
| Wash Buffer | 50mM HEPES, 5mM CaCl2, 0.5% Triton X, pH 8.0 |
| Solubilization Buffer | 50mM HEPES, 5mM CaCl2, 6M guanidinium chloride, pH 8.0 |
| Refolding Buffer | 50mM HEPES, 5mM CaCl2, 4 micromolar ZnCl2 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 48h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | E. coli cultures were centrifuged at 4,000 ?g for 15 min, the pellet resuspended in 50 mM Hepes pH 8.0 containing 5 mM CaCl2, and the cells lysed by sonication on ice. The lysate was centrifuged at 12,000 ?g for 20 min, and the pellet was washed by sonicating in the same buffer containing 0.5% (v/v) Triton X-100. After recentrifugation, the pellet was dissolved in 50 mM Hepes pH 8.0, containing 6 M Gdm-HCl and 5 mM CaCl2, and the preparation was centrifuged at 70,000 ?g for 30 min to remove insoluble material. The supernatants were incubated with the Co2+ resin, previously equilibrated in the solubilization buffer for 20 min at room temperature, and the resin was extensively washed with the buffer containing 0.5 M NaCl. For naturation experiments, aliquots of the resin were dialyzed against 50 mM Hepes pH 7.0 containing 5 mM CaCl2 and 4 µM ZnCl2for 48 h at 4 °C. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | |