Refolding Record:
| Protein | |
|---|---|
| Protein Name | AMP-Forming acetyl-CoA synthetase |
| Abbreviated Name | ACS |
| SCOP Family | Acetyl-CoA synthase |
| Structure Notes | |
| Organism | Haloarcula marismortui |
| UniProt Accession | Q5V2H9 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Multi-domain proteins (alpha and beta) |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 686 |
| Molecular Weight | 76784.6 |
| Pi | 4.1 |
| Molecular Weight | 76784.6 |
| Disulphides | 0 |
| Full Sequence |
MSDEDVQLEARLEEQEVFEPPESFVEQANVTDEGIYDEFEENWPECWEGAADLLDWEEEY
DQVLDDSNPPFYEWFTDGTLNASANCLDRHLDERGDEAAIEWVGEPVEEDNITYTYEELH
QKVNEFAAGLREMGVGEDDVVTMYMPMIPQLPIAMLACARIGAPHSVVFAGFSADALATR
MNSADSEYLVTCDGYYRRGDPLDHLDKANEGLSGVDHEVERAIVAERLMDGDGFDHDYAD
NQVAFEDVIDDNEGETVEPVDRDAEDMLFLMYTSGTTGKPKGVKHSTGGYLAWAAWTSQA
VLDIKPEDTYFCSADIGWITGHSYIVYGPLALGTTTMMYEGTPDYPDKDRLWDIVEEYEA
DQLYTAPTAIRAFMKWGKQYPEQHDLSSLRLLGTVGEPINPRAWKWYYKHIGNEECPVVD
TWWQTETGGMMITTLPGVKDMKPGSAGPPLPGNDVRIVDTEGEEVEPGRAGYLTVDKPWP
GMLRTLYKNDERFIDEYWAEYSDTDSDDSDDWVYFPEDGAKIDDDGYITVLGRVDDVINV
SGHRLGTMEIESAIVGVEGVAEAAVVGGDHDIKGEAVYAYVITEDGYDEDEELRSAIIDG
VEDAIGPIARPEAVIFTPELPKTRSGKIMRRLLEDIANGEELGDTSTLRNPDVVSDIETK
VQGDENIRLLTKPERKSYLLPPLSDD
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Extremophiles (2005) Eur J Biochem., 9, 355-365 |
| Project Aim | Recombinant Protein Expression,Evolutionary Studies |
| Fusion | C-terminal sequence |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)CodonPlus-RIL |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pET17b |
| Expression Protocol | Cells, grown in LB medium, were induced, incubated and then harvested by centrifugation. Cells were then resuspended in Buffer A (20 mM TrisHCl, pH 7.5, 2 M KCl, 2 mM EDTA) and treated with 100 microg/ml lysozyme and 0.1 (v/v) Triton X-100 and incubated for 1h at 30min before transferring the solution onto ice. The suspension was then sonicated and centrifuged(40,000g, 30min, 4degC). The resulting pellet was washed 2x in Buffer A and recentrifuged. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 20 mM TrisHCl, pH 7.4, 8 urea, 2mM EDTA, 50 mM DTE |
| Refolding Buffer | 20 mM TrisHCl, pH 7.5, 2 M KCl, 2 mM EDTA 2 mM ATP, 2.5 mM sodium acetate, 10 microM CoA, 3 mM GSH, 0.3 mM GSSG |
| 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 | GSH/GSSG |
| Redox Agent Concentration | 0.3 mM |
| Refolding Protocol | Inclusion bodies were solubilized, in solubilization buffer, and were refolded by gradual dilution into refolding buffer such that the final concentration of protein was equal to 0.03 mg/ml. Following this the refolding solution was incubated for 1h at 4degC. After refolding, the protein was concentrated by ultrafiltration and adjusted to 2 M (NH4)2SO4 and the solution was centrifuged (100,000g, 45min, 4degC). Supernatant was then applied to a Phenyl-Sepharose 6ff column equilibrated with Buffer B (50mM TrisHCl, pH 7.5, 20mM MgCl2, and 2M (NH4)2SO4. Protein was desorbed with Buffer C (50mM TrisHCl, pH 7.5, 20% glycerol with a decreasing gradient 2-0 M of (NH4)2SO4. |
| Refolding Assay | Ligand Binding,Sequence Analysis,Stability Determination |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |