Refolding Record:
| Protein | |
|---|---|
| Protein Name | Monogalactosyldiacylglycerol synthase |
| Abbreviated Name | MGDG synthase |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Spinacia oleracea |
| UniProt Accession | Q9SM44 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 530 |
| Molecular Weight | 57511.2 |
| Pi | 9.15861 |
| Molecular Weight | 57511.2 |
| Disulphides | Unknown |
| Full Sequence |
MSHPSTVTSEPSNLLDFVPKLGNFVLNSSLHGNNSNGYSSFSSNSVHFGGLATQNRYKFV
NSLSFSKEGSNLKRILSDFNRVIRLHCDRIPLGFSSIGLNSGESNGVSDNGHGVLEDVRV
PVNAVEPESPKRVLILMSDTGGGHRASAEAIKAAFNEEFGDDYQVFVTDLWSEHTPWPFN
QLPRSYNFLVKHGPLWKMMYYGTSPRVIHQSNFAATSVFIAREVARGLMKYQPDIIISVH
PLMQHVPLRILRGRGLLEKIVFTTVVTDLSTCHPTWFHKLVTRCYCPSNEVAKRATKAGL
QPSQIKVYGLPVRPSFVRSVRPKNELRKELGMDEHLPAVLLMGGGEGMGPIEATARALGN
ALYDANLGEPTGQLLVICGRNKKLAGKLSSIDWKIPVQVKGFVTKIEECMGACDCIITKA
GPGTIAEAMIRGLPIILNDYIAGQEAGNVPYVIENGIGKYLKSPKEIAKTVSQWFGPKAN
ELQIMSQNALKHARPDAVFKIVHDLDELVRQKIFVRQYSCAA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Nishiyama Y, Hardre-Lienard H, Miras S, Miege C, Block MA, Revah F, Joyard J, Marechal E. (2003) Protein Expression and Purification, 31, 79-87 |
| Project Aim | Crystallography |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pET-y3a |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication/French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Solubility | not stated |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 40mM Tris-HCl, 10mM EDTA, 0.5% Triton X-100, pH 7.0 |
| Solubilization Buffer | 50mM Mops-NaOH, 10mM DTT, 8M urea, pH 6.5 |
| Refolding Buffer | 0.3M CHAPS, 250mM KCl, 20mM MgCl2, 1mM EDTA, 10mM DTT, 100mM ammonium acetate, 10% glycerol, 10mM Tris-HCl, 50microgram/ml phosphotidylglycerol, 50 microgram/ml 1,2-dioleoyl-sn-glycerol |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 6.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | The target protein was purified from the solubilized inclusion bodies by ion exchange chromatography under denaturing condiitons (8M urea) and subsequently by size exclusion chromatography in 6M guanidinium chloride, 1mM DTT, and 500mM Tris-HCl, pH 7.0. The protein was refolded by step-wise dilution with 0.3M CHAPS, 250mM KCl, 20mM MgCl2, 1mM EDTA, 10mM DTT, 100mM ammonium acetate, 10% glycerol, 10mM Tris-HCl, 50microgram/ml phosphotidylglycerol, 50 microgram/ml 1,2-dioleoyl-sn-glycerol at pH 6.0. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 70-90% |
| Purity | |
| Notes | |