Refolding Record:
| Protein | |
|---|---|
| Protein Name | Dihydrolipoyl dehydrogenase, mitochondrial |
| Abbreviated Name | L-protein |
| SCOP Family | FAD/NAD-linked reductases, N-terminal and central domains |
| Structure Notes | |
| Organism | Pisum sativum (Garden pea) |
| UniProt Accession | P31023 |
| 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 | 471 |
| Molecular Weight | 49744.1 |
| Pi | 6.05 |
| Molecular Weight | 49744.1 |
| Disulphides | 1 |
| Full Sequence |
ASGSDENDVVIIGGGPGGYVAAIKAAQLGFKTTCIEKRGALGGTCLNVGCIPSKALLHSSHMYHEAKHSFANHGVKVSNVEIDLAAMMGQKDKAVSNLTRGIEGLFKKNKVTYVKGYGKFVSPSEISVDTIEGENTVVKGKHIIIATGSDVKSLPGVTIDEKKIVSSTGALALSEIPKKLVVIGAGYIGLEMGSVWGRIGSEVTVVEFASEIVPTMDAEIRKQFQRSLEKQGMKFKLKTKVVGVDTSGDGVKLTVEPSAGGEQTIIEADVVLVSAGRTPFTSGLNLDKIGVETDKLGRILVNERFSTNVSGVYAIGDVIPGPMLAHKAEEDGVACVEYLAGKVGHVDYDKVPGVVYTNPEVASVGKTEEQVKETGVEYRVGKFPFMANSRAKAIDNAEGLVKIIAEKETDKILGVHIMAPNAGELIHEAAIALQYDASSEDIARVCHAHPTMSEAIKEAAMATYDKPIHI
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Faure M, Bourguignon J, Neuburger M, MacHerel D, Sieker L, Ober R, Kahn R, Cohen-Addad C, Douce R. (2000) Eur J Biochem., 267, 2890-2898 |
| Project Aim | Structural Studies |
| 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 | pET-LM |
| Expression Protocol | The expression plasmids described above were transformed into expression hosts E. coli BL21(DE3) or B834 (DE3)pLysS. Culture conditions and target gene expression were performed essentially according to the pET system manual (Novagen, Madison WI, USA). Cells harbouring pET-LM were grown under agitation at 37 °C in 4 × 2-L flasks containing each 600 mL Luria--Bertani medium with carbenicillin (150 µg·mL−1) to a D600 of 0.5. Induction of the L-protein expression was started by addition of isopropyl thio-β-d-galactoside at the final concentration of 0.4 mm and after 3 h, the suspension was cooled on ice and centrifuged at 4 °C (4000 g, 15 min). All subsequent operations were carried out at 4 °C. The cell pellets were suspended in 70 mL of buffer containing 40 mm Tris pH 7.5 and 10 mm EDTA (Tris/EDTA buffer) frozen in liquid nitrogen and stored at −80 °C. After thawing, lysozyme was added to the bacterial suspension to a final concentration of 10 µg·mL−1. The cells were then stored for 30 min at 30 °C, sonicated using an Ultrasons Annemasse apparatus (5 × 1 min, 200 V) and centrifugated (35 000 g, 15 min). The pellets, which contained the overexpressed L-protein present in inclusion bodies, were resuspended in 400 mL of solution containing 40 mm Tris pH 7.5, 10 mm EDTA and 0.5% Triton X100 (Tris/EDTA/Triton buffer), and inclusion bodies were sedimented by centrifugation (11 000 g, 10 min). The inclusion bodies were washed three other times with Tris/EDTA/Triton buffer, once with Tris/EDTA buffer and stored as a pellet at −80 °C. |
| 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 | 40 mm Tris pH 7.5, 10 mm EDTA and 0.5% Triton X100 (Tris/EDTA/Triton buffer) |
| Solubilization Buffer | 2 mL of 6 m guanidine hydrochloride, 50 mm Tris (pH 8.4) and 100 mm 1,4-dithiothreitol |
| Refolding Buffer | 100 mm Tris (pH 8.4), 5 mm EDTA, 20% (v/v) glycerol and 20 µm FAD |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.4 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 200 mg |
| Refolding Time | 30 min |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Refolding from inclusion bodies and purification of the recombinant L-protein The proteins present in the inclusion bodies were resuspended with 2 mL of 6 m guanidine hydrochloride, 50 mm Tris (pH 8.4) and 100 mm 1,4-dithiothreitol and incubated for 1 h at 30 °C. Different refolding conditions were tried as described in [21] and one condition was found for the renaturation of the L-protein. This was performed at room temperature by diluting slowly the protein solution (2.45 mL, around 200 mg of protein) with 400 mL of 100 mm Tris (pH 8.4), 5 mm EDTA, 20% (v/v) glycerol and 20 µm FAD. After 30 min, residual aggregates were removed by centrifugation (11 000 g, 15 min) and the supernatant containing the folded L-protein was concentrated to 10–15 mL by ultrafiltration using a 400-mL stirred cell (Amicon) equipped with an Omega disc membrane (30 K, Ø 76 mm, Pall Filtron). The protein solution was then diluted again to 50 mL with 20 mm KH2PO4 (pH 7.2) and concentrated to a volume of 4–5 mL by ultrafiltration on a 30 K Omega membrane (Pall Filtron) using a 50-mL-stirred cell (Amicon). The protein solution was then incubated at 30 °C for 20 min in presence of RNase A (10 µg·mL−1), DNase I (10 µg·mL−1) and 1 mm MgCl2 and filtered through a 0.2-µm filter. The protein solution was then applied to a Mono Q HR 10/10 column previously equilibrated with a buffer A containing 20 mm KH2PO4 (pH 7.2), 1 mm EDTA and 0.5 mm 1,4-dithiothreitol. The proteins were eluted with a continuously increasing KH2PO4 gradient (20–500 mm in 1 h, flow rate 1 mL·min−1, fraction size 1 mL). The fractions containing the L-protein eluted at the concentration around 300 mm KH2PO4 were combined, diluted with buffer A and concentrated by ultrafiltration. Generally 20 mg of purified L-protein were obtained at the end of the purification. Analysis by nondenaturant polyacrylamide gel electrophoresis confirmed that the recombinant L-protein is a dimer as the native protein. The L-protein (DLDH) activity was assayed according to Neuburger et al. [13] using TCEP as a reducing agent. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 20% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |