Refolding Record:
| Protein | |
|---|---|
| Protein Name | Rhoptry protein |
| Abbreviated Name | ROP2 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Toxoplasma gondii |
| UniProt Accession | Q06AK3 |
| 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 | 257 |
| Molecular Weight | 29548.3 |
| Pi | 9.12 |
| Molecular Weight | 29548.3 |
| Disulphides | Unknown |
| Full Sequence |
LRLLRGIKNQKQAKVHLRFIFPFDLVKDPKKRKMIRVRLDERDMWVLSRFFLYPRMQSNLQMLGDVLLSHSSTHKSLVHHARLQLTLQLIRLLASLHHYGLVHADFQVSNILLDQRGGVFLTGFEHLVRDGASAVSPIGRGFAPPETTAERMLPFGQHHPTLMTFAFDTWTLGLAIYWIWCADLPNTDDAALGGSEWIFRSCKNIPQPVRALLEGFLRYPKEDRLLPLQAMETPEYEQLRTELSAVLPLYQTDGEPA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Jacquet A, Daminet V, Haumont M, Garcia L, Chaudoir S, Bollen A, Biemans R. (1999) Protein Expression and Purification, 17, 392-400 |
| Project Aim | Expression and charactrization |
| Fusion | C-terminal maltose-binding protein |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | G1724 |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pNIV4736 |
| Expression Protocol | E. coli containing the recombinant expression vector pNIV4735 was grown overnight at 37°C in 869 medium with 100 g/ml ampicillin. Cells were then diluted 1:100 and allowed to grow at 37°C to an optical density between 0.4 and 0.6 at 600 nm. Isopropyl B-D-thioga-lactoside (IPTG) was added to a final concentration of 0.3 mM. Following another 4 h of growth, cells were harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.4-0.6 = 600 |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 20 mM Tris–HCl, pH 7.5 |
| Solubilization Buffer | 20 mM Tris–HCl containing 8 M urea |
| Refolding Buffer | 50 mM Tris–HCl, pH 7.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Purification of the insoluble form. The pellet resulting from the ultracentrifugation was washed with 20 mM Tris–HCl, pH 7.5. Insoluble MBP–recROP2t was extracted overnight at 4°C with 20 mM Tris–HCl containing 8 M urea. The suspension was ultracentrifugated at 150,000g for 60 min. The supernatant was directly dialyzed against 50 mM Tris–HCl, pH 7.5. The solution was centrifuged to remove any precipitated protein. Renatured MBP–recROP2t was purified onto an amylose resin as previously described, the eluted peak was concentrated. |
| Refolding Assay | Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |