Refolding Record:
| Protein | |
|---|---|
| Protein Name | Tubulin beta chain |
| Abbreviated Name | Beta-tubulin |
| SCOP Family | Tubulin, GTPase domain |
| Structure Notes | |
| Organism | Haemonchus contortus (Barber pole worm) |
| UniProt Accession | Q25022 |
| 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 | 448 |
| Molecular Weight | 49842.1 |
| Pi | 4.81 |
| Molecular Weight | 49842.1 |
| Disulphides | Unknown |
| Full Sequence |
MREIVHVQAGQCGNQIGAKFWEVISDEHGIQPDGSYKGESDLQLERINVYYNEANGGKYVPRAVLVDLEPGTMDSVRSGPFGALFRPDNFVFGQSGAGNNWAKGHYTEGAELVDSVLDVVRKEAEGCDCLQGFQLTHSLGGGTGSGMGTLLIAKIREEYPDRIMSSFSVVPSPKVSDTVVEPYNATLSVHQLVENTDETFCIDNEALYDICFRTLKLTNPTYGDLNHLVSVTMSGVTTCLRFPGQLNADLRKLAVNMVPFPRLHFFMPGFAPLSAKGAQAYRALTVSELTQQMFDANNMMAACDPRHGRYLTVAAMFRGRMSMREVDDQMMSVQNKNSSYFVEWIPNNVKTAVCDIPPRGLKMAATFVGNSTAIQELFKRISEQFTAMFRRKAFLHWYTGQGMDEMEFTEAESNMNDLVSEYQQYQEATADDEGEMEGAVENDTYAEE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | W. Lubega G, G. Geary T, D. Klein R and K. Prichard R (1993) Molecular and Biochemical Parasitology, 62, 281-292 |
| Project Aim | Over expression & Renaturation |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | Jm101 |
| Expression Temp | 37.0 |
| Expression Time | 1 h |
| Expression Vector | pTrp2 |
| Expression Protocol | Induction of gene expression. The Ptrp promoter was induced under tryptophan starvation conditions [30]. A single positive colony was grown at 37°C to mid-log phase (A600=0.8-1.0) in 500 ml of LB medium containing ampicillin (100 ug /ml ) and tryptophan (Sigma) (1 mg m1-1) to repress promoter activity. To induce expression by tryptophan starvation, the grown cells were collected by centrifugation under aseptic conditions and cultured for a further 1 h at 37°C in 1 1 of M9 minimal medium containing glucose (6 mg ml-l), ampicillin (100 ug/ ml) and 0.1% acid-hydrolysed casein amino acids (Sigma). |
| Method of Induction | Tryptophan starvation |
| Cell Density at Induction | OD 0.8-1.0 = 600 |
| Cell Disruption Method | None |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 0.5% Triton X-100 and 10 mM EDTA |
| Solubilization Buffer | 3 ml of 8 M urea |
| Refolding Buffer | 50 mM KH2PO4 (pH 10.7) containing 0.1 mM PMSF, 1 mM EDTA and 50 mM NaC1 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 10.7 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | 0.1 mM,n/a,0.1 mM |
| Refolding Protocol | Solubilization of the inclusion bodies and renaturation of tubulin. Inclusion bodies were solubilized and renatured utilizing the Urea-Alkaline method [32]. The pellet was dissolved in 3 ml of 8 M urea and incubated for 1 h at room temperature, diluted 20 times with 50 mM KH2PO4 (pH 10.7) containing 0.1 mM PMSF, 1 mM EDTA and 50 mM NaC1 and stored for 30 rain at room temperature. The pH was then adjusted to 8.0 and the incubation continued for a further 30 min. The sample was centrifuged at 12 000xg for 30 min and the supernatant concentrated to a third of its original volume utilising Amicon cones (CF50A) as described by the supplier (Amicon, ON). The concentrate was then diluted 3 times with MES buffer (pH 6.5) consisting of 0.025 M MES, 1 mM EGTA, 0.5 mM MgSO4 and 1 mM GTP and re-concentrated to one third. This was diluted again with MES buffer and concentrated twice. The sample was centrifuged (100 000 xg, 1 h) at 4°C to ensure that there was no aggregated tubulin. Protein concentration was measured by the method of Bradford [33] using purified vertebrate brain tubulin as standard. PMSF (0.1 mM) was added and the recombinant tubulin stored in liquid nitrogen. Tubulin was diluted to 0.1 ug/ul with MES buffer and stored on ice for at least 30 min before use. For comparison, samples containing B 8-9 and B l2 16 were processed simultaneously. |
| Refolding Assay | Western Blot,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |