Refolding Record:
| Protein | |
|---|---|
| Protein Name | Tubulin beta chain |
| Abbreviated Name | Beta-tubulin |
| SCOP Family | Tubulin, GTPase domain |
| Structure Notes | |
| Organism | Trypanosoma brucei |
| UniProt Accession | P04107 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 442 |
| Molecular Weight | 49704.1 |
| Pi | 4.72 |
| Molecular Weight | 49704.1 |
| Disulphides | Unknown |
| Full Sequence |
MREIVCVQAGQCGNQIGSKFWEVISDEHGVDPTGTYQGDSDLQLERINVYFDEATGGRYVPRSVLIDLEPGTMDSVRAGPYGQIFRPDNFIFGQSGAGNNWAKGHYTEGAELIDSVLDVCCKEAESCDCLQGFQICHSLGGGTGSGMGTLLISKLREQYPDRIMMTFSIIPSPKVSDTVVEPYNTTLSVHQLVENSDESMCIDNEALYDICFRTLKLTTPTFGDLNHLVSAVVSGVTCCLRFPGQLNSDLRKLAVNLVPFPRLHFFMMGFAPLTSRGSQQYRGLSVPELTQQMFDAKNMMQAADPRHGRYLTASALFRGRMSTKEVDEQMLNVQNKNSSYFIEWIPNNIKSSVCDIPPKGLKMAVTFIGNNTCIQEMFRRVGEQFTLMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVSEYQQYQDATIEEEGEFDEEEQY
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | W. Lubega G, O. K. Ochola D and K. Prichard1 R (2002) Experimental Parasitology, 102, 134-142 |
| Project Aim | Vaccine studies |
| Fusion | None |
| Protein Expression and Production | Protein expressed and purified in native conformation prior to denaturation and refolding. |
| Expression Host | None |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 0 |
| Expression Vector | |
| Expression Protocol | |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 100 mM PIPES, 1 mM EGTA, 1 mM MgSO4, 1 mM PMSF, pH 6.9 |
| Solubilization Buffer | 3 ml 8 M urea |
| Refolding Buffer | 50 mM KH2 PO4, 0.1 mM PMSF, 1 mM EDTA, and 50 mM NaCl |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 10.7 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | 0.1 mM,n/a |
| Refolding Protocol | Solubilization and renaturation of tubulin The tubulin, purified as above, was solubilized and renatured using a modification (Lubega et al., 1993) of a described procedure (Martson, 1987). Briefly the pellet was dissolved in 3 ml 8 M urea and incubated at 25 °C for 1 h and then diluted about 20 times with alkaline buffer pH 10.7 (50 mM KH2 PO4, 0.1 mM PMSF, 1 mM EDTA, and 50 mM NaCl) and incubated for a further 30 min. The pH was then adjusted to 8.0 and the supernatant was concentrated to one third by ultrafiltration in CF50A membrane cones (Amicon) and re-diluted 3 times with MES buffer (0.025 MES, 1 mM EGTA, 0.5 mM MgSO4, 1 mM GTP, pH 6.0). This cycle of concentration and redilution was repeated three times. The final volume was centrifuged for 2 h at 40,000g, 4 °C to ensure that there was no aggregated tubulin. The purified, renatured tubulin was then stored in liquid nitrogen until needed for immunization and related studies. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | |