Refolding Record:
| Protein | |
|---|---|
| Protein Name | Dehydrodolichyl diphosphate synthase |
| Abbreviated Name | DDPPs |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Yeast (Saccharomyces cerevisiae) |
| UniProt Accession | P35196 |
| 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 | 290 |
| Molecular Weight | 32693.6 |
| Pi | 7.66047 |
| Molecular Weight | 32693.6 |
| Disulphides | Unknown |
| Full Sequence |
METDSGIPGHSFVLKWTKNIFSRTLRASNCVPRHVGFIMDGNRRFARKKEMDVKEGHEAG
FVSMSRILELCYEAGVDTATVFAFSIENFKRSSREVESLMTLARERIRQITERGELACKY
GVRIKIIGDLSLLDKSLLEDVRVAVETTKNNKRATLNICFPYTGREEILHAMKETIVQHK
KGAAIDESTLESHLYTAGVPPLDLLIRTSGVSRLSDFLIWQASSKGVRIELLDCLWPEFG
PIRMAWILLKFSFHKSFLNKEYRLEEGDYDEETNGDPIDLKEKKLN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Chang SY, Tsai PC, Tseng CS, Liang PH. (2001) Protein Expression and Purification, 23, 432-439 |
| Project Aim | Undefined |
| Fusion | N-terminal thioredoxin + hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 30.0 |
| Expression Time | 2h |
| Expression Vector | pET32Xa/LIC |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | unknown |
| Solubilization Buffer | 25 mM Tris (pH 7.5), 150 mM NaCl, 7 M urea |
| Refolding Buffer | 25 mM Tris, 150 mM NaCl, 6 mM b-mercaptoethanol, 0.1% Triton X-100, and 5 mM imidazole |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 1.0mg/ml |
| Refolding Time | |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The pellet collected by centrifugation of the disrupted cells in the absence of detergent was stirred overnight with 50 mL of 25 mM Tris (pH 7.5), 150 mM NaCl, and 7 M urea to dissolve DDPPs. The solution with denatured protein was loaded in a 20-mL Ni?NTA column and washed with the same buffer containing 5 mM imidazole. Protein was then eluted with a 25 mM Tris (pH 7.5), 150 mM NaCl, 7 M urea, and 300 mM imidazole buffer. The purified recombinant protein was refolded on the Ni?NTA column. The protein sample in urea was loaded onto a 20-mL Ni?NTA column. After loading, the column was washed with 5 column vol of buffer containing 25 mM Tris (pH 7.5), 150 mM NaCl, 6 mM b-mercapto- ethanol, 0.1% Triton X-100, and 5 mM imidazole. The bound protein was eluted with solution of 300mMimid- azole in 25 mM Tris (pH 7.5), 150 mM NaCl, 6 mM b-mercaptoethanol, and 0.1% Triton X-100. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 52% |
| Purity | |
| Notes | |