Refolding Record:
| Protein | |
|---|---|
| Protein Name | Streptavidin |
| Abbreviated Name | SCD |
| SCOP Family | Avidin/streptavidin |
| Structure Notes | |
| Organism | Streptomyces avidinii |
| UniProt Accession | P22629 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Tetramer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | W79F |
| Chain Length | 159 |
| Molecular Weight | 16451.9 |
| Pi | 6.1 |
| Molecular Weight | 16451.9 |
| Disulphides | 0 |
| Full Sequence |
DPSKDS KAQVSAAEAG ITGTWYNQLG STFIVTAGAD GALTGTYESA VGNAESRYVL TGRYDSAPAT DGSGTALGWT
VAFKNNYRNA HSATTWSGQY VGGAEARINT QWLLTSGTTE ANAWKSTLVG HDTFTKVKPS AASIDAAKKA GVNNGNPLDA VQQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Chilkoti A, Tan PH, Stayton PS (1995) Proc. Natl. Acad. Sci. USA, 92, 1754-1758 |
| Project Aim | Structure-Function |
| 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 | 37.0 |
| Expression Time | 3h |
| Expression Vector | pET-21a |
| Expression Protocol | 6.5L 2xTY broth with 100microg/ml ampicillin was inoculated with 10ml overnight culture. Cells were grown at 37degC until OD600 reached 1.0, then 1mM IPTG was added. Cells were cultured for a further 3h, then harvested by centrifugation (4500g, 10min) |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 1.0 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50mM TrisHCl pH 8.0, 10mM EDTA, 1.5M NaCl, 1mM PMSF, 0.5% Triton X-100 |
| Solubilization Buffer | 6M GdnHCl pH 1.5 |
| Refolding Buffer | 50mM TrisHCl pH 8.0, 150mM NaCl, 10mM EDTA, 0.1mM PMSF, 0.5mM benzamidineHCl |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 24h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cell pellet was resuspended in 200ml of 50ml Tris pH 8.0, 0.75M sucrose, 1mM PMSF and lysed by sonication, then incubated with 15min with 10microg/ml DNaseI, 10microg/ml RNaseA, 10mM MgCl2. The lysate was centrifuged (22000g, 30min), then washed with wash buffer, once with Triton X-100 then four times without Triton X-100. The inclusion bodies were dissolved in 500ml solubilization buffer to a concentration of less than 50microM, then dialysed against 20L of refolding buffer for 24h with one 20L change of buffer. The dialysate was centrifuged and filtered. The protein was then purified by affinity chromatography with iminobiotin-agarose. |
| Refolding Assay | Ligand Binding,SDS-PAGE,Amino acid sequencing,ELISA |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |