Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cysteine protease 14 |
| Abbreviated Name | Tr-cp14 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Trifolium repens |
| UniProt Accession | Q6Y1E9 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 354 |
| Molecular Weight | 39974.9 |
| Pi | 6.02444 |
| Molecular Weight | 39974.9 |
| Disulphides | Unknown |
| Full Sequence |
MASMTGGQQMGRGS RDFSIV GYSSEDLKSM DKLIELFESW
MSRHGKIYET IEEKLLRFEV FKDNLKHIDE RNKIVSNYWL GLNEFADLSH QEFKNKYLGL KVNLSQRRES SNEEEFTYRD VDLPKSVDWR KKGAVTPVKN QGQCGSCWAF STVAAVEGIN QIVTGNLTSL SEQELIDCDT TYNNGCNGGL MDYAFSFIVQ NGGLHKEDDY PYIMEESTCE MKKEETQVVT INGYHDVPQN NEQSLLKALA NQPLSVAIEA SSRDFQFYSG GVFDGHCGSD LDHGVSAVGY GTSKNLDYII VKNSWGAKWG EKGFIRMKRN IGKPEGICGL YKMASYPTKK K KLAAALEHHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Asp T, Bowra S, Borg S, Holm PB (2004) Biochim Biophys Acta., 1699, 111-122 |
| Project Aim | Structure-Function |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)CodonPlus-RIL |
| Expression Temp | 37.0 |
| Expression Time | 2 |
| Expression Vector | pET21b |
| Expression Protocol | 2.5L E.coli cells were induced with 1mM IPTG and incubated for 2h. Cellswere harvested by centrifugation (6000rpm x 15min, 4degC). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 20mM imidazole, 0.5M NaCl, 40mM TrisHCl pH 7.9, 6M Urea |
| Solubilization Buffer | 5mM imidazole, 0.5M NaCl, 40mM TrisHCl pH 7.9, 6M Urea |
| Refolding Buffer | 50mM K2HPO4 pH 8.0, 5mM EDTA, 1mM reduced glutathione, 0.1mM oxidized glutathione, 250mM L-arginine |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | overnight |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1mM/0.1mM |
| Refolding Protocol | The inclusion bodies were isolated according to the His Bind Kits manual (Novagen). The final pellet of inclusion bodies was resuspended in solubilization buffer then loaded onto a 10ml HisBind resin column (Novagen). The column was washed with 100ml solubilization buffer followed by 60ml wash buffer. The protein was then eluted with 60ml elution buffer (wash buffer containing 1M imidazole). The purified protein was reduced by the addition of DTT (final concentration 10mM) and incubation at 37degC for 1h. The protein was then added dropwise into 10L of refolding buffer and stirred overnight at 4degC. The protein was then centrifuged (15min x 6000rpm, 4degC) and the supernatant concentrated by ultrafiltration. The purified protein was then activated by incubation in 100mM sodium acetate pH 5.0, 2mM EDTA, 20mM L-cystein for 7h at 25degC. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 250mM |
| Refolding Yield | 24% |
| Purity | |
| Notes | |