Refolding Record:
| Protein | |
|---|---|
| Protein Name | 18 kDa cyclophilin, Cyclophilin-18 |
| Abbreviated Name | C-18 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Toxoplasma gondii |
| UniProt Accession | Q26994 |
| 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 | 163 |
| Molecular Weight | 17920.2 |
| Pi | 5.87 |
| Molecular Weight | 17920.2 |
| Disulphides | Unknown |
| Full Sequence |
ENAGVRKAYMDIDIDGEHAGRIILELREDIAPKTVKNFIGLFDKYKGSVFHRIIPDFMIQGGDFENHNGTGGHSIYGRRFDDENFDLKHERGVISMANAGPNTNGSQFFITTVKTEWLDARHVVFGKITTESWPTVQAIEALGGSGGRPSKVAKITDIGLLE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Aliberti J, Valenzuela JG, Carruthers VB, Hieny S, Andersen J, Charest H, Reis e Sousa C, Fairlamb A, Ribeiro JM, Sher A. (2003) Nat Immunol, 4, 485-90 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 37.0 |
| Expression Time | 6 h |
| Expression Vector | n/a |
| Expression Protocol | A 500-ml cell culture containing the C-18 expression vector was grown in LB medium with ampicillin (100 g/ml) at 37 °C, and isopropyl--D-thiogalactopyranoside (IPTG) (1 mM) was added during the final 6 h of incubation. Cells were harvested by centrifugation, the pellet resuspended in Tris-HCl, pH 7.4, 20 mM EDTA, and lysozyme (8 mg/ml) added. After 1 h at room temperature with intermittent shaking, NaCl (0.5 M final) and Triton X-100 (2.5% final) were added, and the sample was incubated at room temperature for an additional 30 min. The cells were then centrifuged and the pellet resuspended in Tris-EDTA-1% Triton solution, after which the mixture was sonicated 3 times for 30 s followed by cooling for 1 min on ice. The sample was centrifuged again, and the whole procedure was repeated 5 times with intermittent washes of the pellet. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | NaCl (0.5 M final) and Triton X-100 |
| Solubilization Buffer | 6 M guanidine-HCl, Tris-HCl, pH 8.0, 2 mM EDTA |
| Refolding Buffer | (0.1 M Tris-HCl, pH 8.0, 0.5 M L-arginine-HCl, 0.9 mM GSSG, and 2 mM EDTA |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 36 h |
| Redox Agent | GSSG |
| Redox Agent Concentration | 0.9 mM |
| Refolding Protocol | The pellet was next solubilized in 6 M guanidine-HCl, Tris-HCl, pH 8.0, 2 mM EDTA, sonicated 3 times for 30 s and incubated for 2 h at room temperature. After centrifugation at 47,800g for 30 min at 4 °C, the supernatant was recovered, dithiorythritol (DTE) was added to a final concentration of 65 mM, and the sample was incubated at room temperature for 2 h. The solution was then added to a refolding buffer (0.1 M Tris-HCl, pH 8.0, 0.5 M L-arginine-HCl, 0.9 mM GSSG, and 2 mM EDTA) and the mixture was incubated for 36 h at 4 °C followed by dialysis against 20 mM Tris-HCl, pH 7.4. Finally, the recombinant protein was purified by reverse phase HPLC using an octadecyl-silica column (Vydac, Hesperia, CA). Fractions of the main peak were pooled and Edman degradation was carried out. The pooled material was homogeneous and yielded the expected N-terminal sequence MENAGVRKAYMDIDIDGE. |
| Refolding Assay | Binding assay |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.5 M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |