Refolding Record:
| Protein | |
|---|---|
| Protein Name | Candidapepsin |
| Abbreviated Name | candidapepsin |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Candida tropicalis |
| UniProt Accession | Q00663 |
| 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 | 376 |
| Molecular Weight | 40559.0 |
| Pi | 4.53111 |
| Molecular Weight | 40559.0 |
| Disulphides | 2 |
| Full Sequence |
LFAQGLTIPD GIEKRTDKVV SLDFTVIRKP FNATAHRLIQ KRSDVPTTLI NEGPSYAADI VVGSNQQKQT VVIDTGSSDL WVVDTDAECQ VTYSGQTNNF CKQEGTFDPS SSSSAQNLNQ
DFSIEYGDLT SSQGSFYKDT VGFGGISIKN QQFADVTTTS VDQGIMGIGF TAVEAGYNLY SNVPVTLKKQ GIINKNAYSC DLNSEDASTG KIIFGGVDNA KYTGTLTALP VTSSVELRVH LGSINFDGTS VSTNADVVLD SGTTITYFSQ STADKFARIV GATWDSRNEI YRLPSCDLSG DAVVNFDQGV KITVPLSELI LKDSDSSICY FGISRNDANI LGDNFLRRAY IVYDLDDKTI SLAQVKYTSS SDISAL
|
| Notes | Full sequence expressed and refolded, including prosequence and protein |
| Expression | |
|---|---|
| Report | Lin X, Tang J, Koelsch G, Monod M, Foundling S (1993) J Biol Chem, 268, 20143-20147 |
| Project Aim | Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pET11a |
| Expression Protocol | no details provided |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Freeze-thaw |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 100mM TrisHCl, 150mM NaCl, 1% Triton X-100 pH 7.4 |
| Solubilization Buffer | 8M urea, 50mM Tris, 1mM glycine, 1mM EDTA, 100mM 2-mercaptoethanol pH 8.0 |
| Refolding Buffer | 20mM Tris pH 7.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.5mg/ml |
| Refolding Time | 48h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were harvested by centrifugation and resuspended in 100mM TrisHCl, 150mM NaCl pH 7.4, and 100mg lysozyme was added. The mixture was stirred at room temperature for 30min and frozen at -20degC overnight. After thawing, 200microL of 1M MgCl2 and 50 Kunitz units of DNase were added. The solution was stirred at room temperature for 30min and then diluted to 250ml with wash buffer. This mixture was further stirred at room temperature for 1h and inclusion bodies were then collected by centrifugation (10000rpm, 20min). The pellet was washed with the same volume of wash buffer, and the final pellet was then dissolved in 20ml solubilization buffer. The mixture was centrifuged (30min, 175000g) and the supernatant diluted with solubilization buffer to a final protein concentration of 0.5mg/ml. The solution was then dialyzed for 48h against three changes of refolding buffer at 4degC with continuous stirring. The protein was then concentrated, centrifuged (175000g, 15min) and passed down a Sephacryl S-300 column followed by an anion-exchange monoQ column, all in refolding buffer (anion exchange used linear gradient of 1M NaCl). |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | |