Refolding Record:
| Protein | |
|---|---|
| Protein Name | D-lactate dehydrogenase |
| Abbreviated Name | LDH |
| SCOP Family | Formate/glycerate dehydrogenases, NAD-domain |
| Structure Notes | |
| Organism | Staphylococcus sp. |
| UniProt Accession | A8Z3B9 |
| 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 | 330 |
| Molecular Weight | 36681.6 |
| Pi | 5.09 |
| Molecular Weight | 36681.6 |
| Disulphides | Unknown |
| Full Sequence |
MTKIMFFGTRDYEKEMALNWGKKNNVEVTTSKELLSSATVDQLKDYDGVTTMQFGKLENDVYPKLESYGIKQIAQRTAGFDMYDLDLAKKHNIVISNVPSYSPETIAEYSVSIALQLVRRFPDIERRVQAHDFTWQAEIMSKPVKNMTVAIIGTGRIGAATAKIYAGFGATITAYDAYPNKDLDFLTYKDSVKEAIKDADIISLHVPANKESYHLFDKAMFDHVKKGAILVNAARGAVINTPDLIAAVNDGTLLGAAIDTYENEAAYFTNDWTNKDIDDKTLLELIEHERILVTPHIAFFSDEAVQNLVEGGLNAALSVINTGTCETRLN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Okuda H, Sakuhana C, Yamamoto R, Mizukami Y, Kawai R, Sumita Y, Koga M, Shirai M, Matsuda K. (2008) Biologycal Chemistry, 1, 1 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein expressed and purified in native conformation prior to denaturation and refolding. |
| Expression Host | None |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 0 |
| Expression Vector | |
| Expression Protocol | |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 4 M guanidine hydrochloride for 1 h at 25˚C |
| Refolding Buffer | 0.5 μM GroEL, 1 μM GroES, 2 mM nucleotide, 10 mM magnesium acetate |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 0.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 5 mM |
| Refolding Protocol | LDH refolding assay LDH [EC 1.1.1.28] from Staphylococcus sp. (Amano Enzyme Inc., Aichi, Japan) partially interacts with GroEL and is a homo-dimeric protein with a subunit molecular mass of 32,000. It catalyzes reversible conversions of pyruvate to lactate and of NADH to NAD+. LDH (10 μM) was denatured with 4 M guanidine hydrochloride for 1 h at 25˚C. The unfolded LDH was diluted 100-fold at 25˚C into refoding buffer containing 0.5 μM GroEL, 1 μM GroES, 2 mM nucleotide, 10 mM magnesium acetate and 5 mM β-mercaptoethanol. LDH activity was assayed at 25˚C by monitoring absorbance at 340 nm in LDH assay solution (100 mM Tris-HCl, pH 7.8, 100 μM NADH and 100 μM sodium pyruvate). |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | GroEL,GroES |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | Note in this paper refolding pH isn't specified. |