Refolding Record:
| Protein | |
|---|---|
| Protein Name | NADP-dependant Ds-threo-isocitrate dehydrogenase |
| Abbreviated Name | ICDH |
| SCOP Family | Dimeric isocitrate & isopropylmalate dehydrogenases |
| Structure Notes | |
| Organism | Haloferax volcanii |
| UniProt Accession | Q8X277 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 419 |
| Molecular Weight | 45837.4 |
| Pi | 4.55599 |
| Molecular Weight | 45837.4 |
| Disulphides | Unknown |
| Full Sequence |
MSYDQIEVPDDGEKITVDEETGELSVPDNPIIPIIHGDGIGTDVGPAAQKVLDAAAEATG
RSVSWMRVYAGSSARDKYDENLPEDTVSAIRNHRVAIKGPLTTPVGAGFRSLNVALRKKL
DLYANVRPTYHLDGVPSPVKNPSAMDMVTFRENTEDVYAGIEWEAGTDEVQKVKEFVEEE
MGADGVIHDGPVGIGIKPITEFGTKRLVREAIEYALENDRPSVTLVHKGNIMKFTEGAFR
DWGYELAEEEFGDVTITEDELWEEYDGKRPEDKVVVKDRIADNMLQQLLTRTANYDVIAT
MNLNGDYMSDAAGAQIGGLGIAPGANFGEGLCLAEPVHGSAPKYAGKDKVNPTAMILSGR
LMFEYMGWKDAGKLIRDTVEKTISDGDVTYDLERQIEGGNKLATSEYADKVVENIKELA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Camacho M, Rodreguez-Arnedo A, Bonete MJ (2002) FEMS Microbiology Letters, 209, 155-160 |
| Project Aim | Recombinant Protein Expression |
| 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 | 3 h |
| Expression Vector | pET3a |
| Expression Protocol | 2.3. Expression of H. volcanii isocitrate dehydrogenase in E. coli PCR techniques were used to amplify a gene fragment corresponding to the structural gene of H. volcanii ICDH with 1260 bp, including the digestion sequences of NdeI and BamHI to facilitate the subcloning of the H. volcanii ICDH gene for expression. Template DNA for PCR was the clone from the ? EMBL3 library. The PCR product was subcloned into plasmid pCR?.1. After transforming E. coli Novablue cells (Novagen) with this construct, plasmid DNA was ligated into expression vector pET-3a (Novagen). The resulting clones were propagated in E. coli Novablue cells prior to transforming E. coli BL21 (DE3) (Novagen), which was used as the expression host. Cultures were grown to an OD600nm at 37°C of 0.5?.0 in Luria–Bertani (LB) medium containing 50 ?g ml?1 ampicillin. Induction of expression was achieved by the addition of isopropyl ?--thiogalactoside (IPTG) to a final concentration of 0.4 mM, and the cultures were shaken at 37°C for 3 h before harvesting cells by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | unknown |
| Solubilization Buffer | 20 mM Tris–HCl pH 8.0, containing 2 mM EDTA, 8 M urea and 50 mM dithiothreitol (DTT) |
| Refolding Buffer | 20 mM Tris–HCl pH 8.0, containing 3 M NaCl, 1 mM EDTA and 10 mM MgCl2 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 20 ug/ml |
| Refolding Time | 24 h |
| Redox Agent | DTT |
| Redox Agent Concentration | 50 mM,50 mM |
| Refolding Protocol | 2.4. Refolding and purification of insoluble recombinant isocitrate dehydrogenase E. coli BL21 (DE3) cell pellets were resuspended in 20 mM Tris–HCl buffer, pH 8.0, containing 2 mM EDTA and 2 M KCl, treated with 100 ?g ml?1 lysozyme and 0.1% (v/v) Triton X-100, and incubated at 30°C for 1 h before transferring the solution onto ice for 15 min. The suspension was then sonicated and centrifuged at 10 000×g for 10 min at 4°C. The insoluble fraction yielded inclusion bodies together with the insoluble cell fragments, and it was dissolved in solubilisation buffer (20 mM Tris–HCl pH 8.0, containing 2 mM EDTA, 8 M urea and 50 mM dithiothreitol (DTT)). Refolding was initiated by slowly diluting the solubilised pellet to a final concentration of 20 ?g ml?1 into renaturation buffer and incubating for at least 24 h at room temperature. The renaturation buffer was composed of 20 mM Tris–HCl pH 8.0, containing 3 M NaCl, 1 mM EDTA and 10 mM MgCl2. The solution was concentrated by ultrafiltration on a PM 50 membrane with a stirred cell (Amicon Inc., Beverly, MA, USA) to a final volume of 10 ml. The buffer was exchanged to 20 mM Tris–HCl pH 8.0, containing 2.5 M (NH4)2SO4, 1 mM EDTA and 10 mM MgCl2 prior to further chromatography on a DEAE-cellulose (Amersham Pharmacia Biotech) column equilibrated with the same buffer, and the enzyme was eluted with the renaturation buffer. Fractions containing isocitrate dehydrogenase activity were pooled and, finally, dialysed against the same renaturation buffer for 24 h at 4°C to remove the (NH4)2SO4 remaining, and stored at this temperature. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |