Refolding Record:
| Protein | |
|---|---|
| Protein Name | Interleukin-1 beta-converting enzyme |
| Abbreviated Name | ICE |
| SCOP Family | Caspase catalytic domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P29466 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Heterodimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 410 |
| Molecular Weight | 45158.6 |
| Pi | 5.63287 |
| Molecular Weight | 45158.6 |
| Disulphides | 1 |
| Full Sequence |
MADKVLKEKRKLFIRSMGEGTINGLLDELLQTRVLNKEEMEKVKRENATVMDKTRALIDS
VIPKGAQACQICITYICEEDSYLAGTLGLSADQTSGNYLNMQDSQGVLSSFPAPQAVQDN
PAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTRLALIICNEEFDSIPRRT
GAEVDITGMTMLLQNLGYSVDVKKNLTASDMTTELEAFAHRPEHKTSDSTFLVFMSHGIR
EGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRGDSPGVVWFKDSVG
VSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPTMGSVFIGRLIEHMQEY
ACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTLTRCFYLFPGH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Ramage P, Cheneval D, Chvei M, Graff P, Hemmig R, Heng R, Kocher HP, Mackenzie A, Memmert K, Revesz L, Wishart W (1995) J Biol Chem, 270, 9378-9383 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | SG936pCI857 |
| Expression Temp | 42.0 |
| Expression Time | 4h |
| Expression Vector | pGI-lambda-PBR#13 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | Reverse phase chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 50 m M Tris, pH 8.0; containing 2 m M DTT, 5 m M benzamidine-HCl, and 2 m M EDTA |
| Solubilization Buffer | 50 m M Tris, pH 8.0, 8 M urea, 50 m M DTT |
| Refolding Buffer | 50 m M Tris, 10 m M DTT |
| Pre-Refolding Purification | Reverse phase chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 32h |
| Redox Agent | GSH |
| Redox Agent Concentration | n/a |
| Refolding Protocol | E. coli wet cell pellets were suspended to 12.5% (w/v) in cell lysis buffer (50 m M Tris, pH 8.0; containing 2 m M DTT, 5 m M benzamidine-HCl, and 2 m M EDTA) and mixed by stirring for 1 h on ice. The cell suspension was lysed by passage through a Manton-Gaulin homogenizer (2 passes at 1200 bar) and then centrifuged for 30 min at 16,000 g. The resultant pellets were resuspended in lysis buffer and recentrifuged a further 3 times. Semipurified inclusion bodies were solubilized in 6 M guanidine-HCl, 25 m M DTT and purified by sequential preparative reversed phase HPLC on two Orpegen HD gel RP-7 s C8 (22 250 mm) columns using a water/acetonitrile buffer system. p45 containing fractions from the second Orpegen column were pooled and, following removal of organic solvent, lyophilized and stored at -20 °C. Refolding of p45 Lyophilized inclusion bodies were solubilized (1 mg/ml) in 50 m M Tris, pH 8.0 (containing 8 M urea, 50 m M DTT), for 1 h at room temperature. Following dilution with 3 volumes of 50 m M Tris, pH 8.0, 10 m M DTT, the refolding mixture was stirred overnight at room temperature and then dialyzed overnight at 4 °C against 2 80 volumes of 50 m M Tris, pH 8.0, containing 10 m M GSH. Upon dialysis and disulphide formation, the 45kDa protein self-processed to the correct 10kDa and 20kDa forms. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | more than 80% |
| Notes | |