Refolding Record:
| Protein | |
|---|---|
| Protein Name | Tailspike Protein |
| Abbreviated Name | TSP |
| SCOP Family | P22 Tailspike Protein |
| Structure Notes | |
| Organism | Bacteriophage P22 |
| UniProt Accession | P12528 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Trimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 667 |
| Molecular Weight | 71856.9 |
| Pi | 5.34 |
| Molecular Weight | 71856.9 |
| Disulphides | 0 |
| Full Sequence |
MTDITANVVVSNPRPIFTESRSFKAVANGKIYIGQIDTDPVNPANQIPVYIENEDGSHVQ
ITQPLIINAAGKIVYNGQLVKIVTVQGHSMAIYDANGSQVDYIANVLKYDPDQYSIEADK
KFKYSVKLSDYPTLQDAASAAVDGLLIDRDYNFYGGETVDFGGKVLTIECKAKFIGDGNL
IFTKLGKGSRIAGVFMESTTTPWVIKPWTDDNQWLTDAAAVVATLKQSKTDGYQPTVSDY
VKFPGIETLLPPNAKGQNITSTLEIRECIGVEVHRASGLMAGFLFRGCHFCKMVDANNPS
GGKDGIITFENLSGDWGKGNYVIGGRTSYGSVSSAQFLRNNGGFERDGGVIGFTSYRAGE
SGVKTWQGTVGSTTSRNYNLQFRDSVVIYPVWDGFDLGADTDMNPELDRPGDYPITQYPL
HQLPLNHLIDNLLVRGALGVGFGMDGKGMYVSNITVEDCAGSGAYLLTHESVFTNIAIID
TNTKDFQANQIYISGACRVNGLRLIGIRSTDGQGLTIDAPNSTVSGITGMVDPSRINVAN
LAEEGLGNIRANSFGYDSAAIKLRIHKLSKTLDSGALYSHINGGAGSGSAYTQLTAISGS
TPDAVSLKVNHKDCRGAEIPFVPDIASDDFIKDSSCFLPYWENNSTSLKALVKKPNGELV
RLTLATL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Betts, S., Haase-Pettingell, C., Cook, K., King, J. (2004) Protein Science, 13, 2291-2303 |
| Project Aim | Folding |
| 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 | 30.0 |
| Expression Time | 2h |
| Expression Vector | pET11a |
| Expression Protocol | Cells were grown overnight in LB broth supplemented with 50microg/ml ampicillin with shaking. Cells were induced incubated and then harvested by centrifugation (8,000g, 7min, 4degC). Cells were then resuspended in ice-cold lysis buffer (50mM TrisHCl, pH 8.0, 100mM NaCl, 2mM EDTA) and stored at -20degC. Cells were then thawed at room temp. and immediately supplemented with PMSF and DTT to final concentrations of 1mM and 2mM respectively. Lysis was initiated by the addition of 100microg/ml of lysozyme. This solution was incubated at room temperature for 30min with occasional mixing prior to the addition of DNAse and then incubated for a further 20-30min. Inclusion bodies were separated by centrifugation (17,000g, 5min, 4degC). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Freeze-thaw |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M urea, 50 mM TrisHCl, pH 7.6, 25 mM NaCl, 2 mM EDTA, 10 mM DTT |
| Refolding Buffer | 50 mM Tris, 25 mM NaCl, 2 mM EDTA, pH 7.6, 10mM DTT |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.6 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 20microg/ml |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 10mM |
| Refolding Protocol | Inclusion bodies were separated by centrifugation (17,000g, 5min, 4degC) Suspensions of detergent-washed inclusion body preparations containing 20microg tail spike chains were pelleted and resuspended in 50 microL of solubilization buffer. This suspension was allowed to incubated for 60min at 20degC before refolding was initiated by dilution with 950 microL of ice-cold dilution buffer. This was then allowed to incubated for 60min on ice. |
| Refolding Assay | SDS-PAGE,Gel filtration chromatography,Laser densitometry |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |