Refolding Record:
| Protein | |
|---|---|
| Protein Name | Phosphate carrier protein, mitochondrial |
| Abbreviated Name | PiC |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Bovine |
| UniProt Accession | P12234 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 314 |
| Molecular Weight | 35020.0 |
| Pi | 9.23 |
| Molecular Weight | 35020.0 |
| Disulphides | Unknown |
| Full Sequence |
AVEEQYSCDYGSGRFFILCGLGGIISCGTTHTALVPLDLVKCRMQVDPQKYKSIFNGFSVTLKEDGFRGLAKGWAPTFIGYSLQGLCKFGFYEVFKVLYSNMLGEENAYLWRTSLYLAASASAEFFADIALAPMEAAKVRIQTQPGYANTLRDAAPKMYKEEGLKAFYKGVAPLWMRQIPYTMMKFACFERTVEALYKFVVPKPRSECSKPEQLVVTFVAGYIAGVFCAIVSHPADSVVSVLNKEKGSSAS
EVLKRLGFRGVWKGLFARIIMIGTLTALQWFIYDSVKVYFRLPRPPPPEMPESLKKKLGYTQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Fiermonte G, Dolce V, Palmieri F. (1998) Biologycal Chemistry, 273, 22782-22787 |
| Project Aim | Identification and Characterization |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | pET21b |
| Expression Protocol | Bacterial Expression and Purification of the Bovine PiC Isoforms-- The expression of PiC-A and PiC-B as inclusion bodies in the bacterial cytosol was accomplished in Escherichia coli BL21(DE3), as described first for the bovine oxoglutarate carrier (18) and then for several other mitochondrial carriers (11, 12, 19-21). Control cultures containing the empty pET21b vector were processed in parallel. Purified inclusion bodies (18) |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 10 mM Tris-HCl and 0.1 mM EDTA, pH 8.0 |
| Solubilization Buffer | 1.67% (w/v) N-dodecanoylsarcosine (Sarkosyl) |
| Refolding Buffer | 0.1% Sarkosyl, 0.5 M NaCl, and 20 mM Pi, pH 8.0 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 10 min |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | suspended in TE buffer (10 mM Tris-HCl and 0.1 mM EDTA, pH 8.0), were solubilized in 1.67% (w/v) N-dodecanoylsarcosine (Sarkosyl) for 5 min at 0 °C. The solution was diluted 20 times with SSP buffer consisting of 0.1% Sarkosyl, 0.5 M NaCl, and 20 mM Pi, pH 8.0, and centrifuged at 12,000 × g for 10 min at 4 °C. The supernatant was chromatographed on Ni+-nitrilotriacetic acid-agarose affinity column (Quiagen). Unspecifically bound proteins were washed with the SSP buffer supplemented with increasing concentrations of histidine. Pure PiC isoforms A and B were recovered when histidine reached 10 mM. The purified proteins were desalted by a Sephadex G-25 column (PD-10 Pharmacia) and stored at 70 °C. All chromatographic steps were performed at 4 °C. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |