Refolding Record:
| Protein | |
|---|---|
| Protein Name | Trypsin |
| Abbreviated Name | n/a |
| SCOP Family | Eukaryotic Proteases |
| Structure Notes | |
| Organism | Bovine |
| UniProt Accession | P00760 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 224 |
| Molecular Weight | 23305.3 |
| Pi | 8.69 |
| Molecular Weight | 23305.3 |
| Disulphides | 6 |
| Full Sequence |
IVGGYTCGANTVPYQVSLNSGYHFCGGSLINSQWVVSAAHCYKSGIQVRLGEDNINVVEGNEQFISASKSIVHPSYNSNTLNNDIMLIKLKSAASLNSRVASISLPTSCASAGTQCLISGWGNTKSSGTSYPDVLKCLKAPILSDSSCKSAYPGQITSNMFCAGYLEGGKDSCQGDSGGPVVCSGKLQGIVSWGSGCAQKNKPGVYTKVCNYVSWIKQTIASN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Ohshima Y, Suzuki Y, Nakatani A, Nohara D. (2008) J Biosci Bioeng, 106, 345-9 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein expressed and purified in native conformation prior to denaturation and refolding. |
| Expression Host | |
| Expression Strain | |
| Expression Temp | 0.0 |
| Expression Time | 0 |
| Expression Vector | |
| Expression Protocol | |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M guanidinium chloride (GdmCl) at pH 2.0 |
| Refolding Buffer | 1 M diethanolamine pH 8.2, 3 mM glutathione (GSH) and 1 mM oxidized glutathione (GSSG) |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.2 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 3/1 mM |
| Refolding Protocol | Refolding of S-S intact trypsin was carried out from its unfolded state after denaturation with 6 M GdmCl at pH 2.0. The denatured trypsin was diluted with 1 M diethanolamine or other solutions to a final concentration of 100 nM at pH 8.2. In the case of fully reduced trypsin, the refolding solution also contained 3 mM glutathione (GSH) and 1 mM oxidized glutathione (GSSG). For refolding in the presence of inhibitor-immobilized gels that adsorb reactivated trypsin molecules, an elution procedure was needed to release the adsorbed protein to assay for the recovered activity. |
| Refolding Assay | HPLC,Mass spectrometry |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 86% |
| Purity | n/a |
| Notes | n/a |