Refolding Record:
| Protein | |
|---|---|
| Protein Name | Thermopsin |
| Abbreviated Name | Thermopsin |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Sulfolobus acidocaldarius |
| UniProt Accession | P17118 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 345 |
| Molecular Weight | 37261.9 |
| Pi | 4.26824 |
| Molecular Weight | 37261.9 |
| Disulphides | 0 |
| Full Sequence |
MNFKSICLIILLSALIIPYIPQNIYFFPHRNTTGATISSGLYVNPYLYYTSPPAPAGIAS
FGLYNYSGNVTPYVITTNEMLGYVNITSLLAYNREALRYGVDPYSATLQFNIVLSVNTSN
GVYAYWLQDVGQFQTNKNSLTFIDNVWNLTGSLSTLSSSAITGNGQVASAGGGQTFYYDV
GPSYTYSFPLSYIYIINMSYTSNAVYVWIGYEIIQIGQTEYGTVNYYDKITIYQPNIISA
SLMINGNNYTPNGLYYDAELVWGGGGNGAPTSFNSLNCTLGLYYISNGSITPVPSLYTFG
ADTAEAAYNVYTTMNNGVPIAYNGIENLTILTNNFSVILI
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lin X, Liu M, Tang J (1992) Enzyme Microb. Technol., 14, 696-701 |
| Project Aim | Structure-Function |
| Fusion | N-terminal pepsinogen |
| 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 | 3h |
| Expression Vector | pET-3b |
| Expression Protocol | Not Stated |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Freeze/Thaw+Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM Tris-HCl, 150mM NaCl, 1% Triton X-100, pH 7.4 |
| Solubilization Buffer | 8M urea, 20mM Tris-HCl, 1mM EDTA, 1mM glycine, 100mM betamercaptoethanol, pH 8.0 |
| Refolding Buffer | 10mM Tris-HCl |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 9.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 16h |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The inclusion body pellet was washed once and solubilised in 8M urea, 20mM Tris-HCl, 1mM EDTA, 1mM glycine, 100mM betamercaptoethanol, pH 8.0. The solution was stirred overnight and then clarified by centrifugation. The supernatant was mixed with 9 vols of 8M urea, 50mM CAPS, pH 11.5 and then diluted with active stirring into 100 vols of 10mM Tris, pH 9.0. This solution was stored at room temp for 2h and then at 4C overnight. The solution was concentrated, filtered and mixed with 0.5ml of Na formate, pH 3.2. Glycerol was added to a final conentration of 10% and the solution was heated at 70C for 35 min to initiate cleavage of the pepsinogen tag. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | |