Refolding Record:
| Protein | |
|---|---|
| Protein Name | SAG1 protein also known as P30 |
| Abbreviated Name | SAG1 |
| SCOP Family | Major surface antigen p30, SAG1 |
| Structure Notes | |
| Organism | Toxoplasma gondii |
| UniProt Accession | Q27298 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 337 |
| Molecular Weight | 34761.7 |
| Pi | 7.89 |
| Molecular Weight | 34761.7 |
| Disulphides | Unknown |
| Full Sequence |
MSVSLHHFIISSGFLTSMFPKAVRRAVTAGVFAAPTLMSFLRCGAMASDPPLVANQVVTCPDKKSTAAVILTPTENHFTLKCPKTALTEPPTLAYSPNRQICPAGTTSSCTSKAVTLSSLIPEAEDSWWTGDSASLDTAGIKLTVPIEKFPVTTQTFVVGCIKGDDAQSCMVTVTVQARASSVVNNVARCSYGANSTLGPVKLSAEGPTTMTLVCGKDGVKVPQDNNQYCSGTTLTGCNEKSFKDILPKLSENPWQGNASSDNGATLTINKEAFPAESKSVIIGCTGGSPEKHHCTVQLE
FAGAAGSAKSSAGTASHVSIFAMVTGLIGSIAACVA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kimbita EN, Xuan X, Huang X, Miyazawa T, Fukumoto S, Mishima M, Suzuki H, Sugimoto C, Nagasawa H, Fujisaki K, Suzuki N, Mikami T, Igarashi I. (2001) Veterinary Parasitology, 102, 35-44 |
| Project Aim | Diagnostic studies |
| Fusion | GST |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | DH5α |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | pGEX-4T-3 |
| Expression Protocol | The gene encoding SAG1 of T. gondii occurs as a single copy, without introns and therefore genomic DNA was cloned by methods described by Burg et al. (1988) with some modifications. Briefly, the genomic DNA extracted as described above, was used as a template for polymerase chain reaction (PCR) amplification, which used the primers 5′-ACG AAT TCG ACG AGT ATG TTT-3′ and 5′-ACG AAT TCA ACG GTG ATC-3′ together with PCR buffer, dNTP and Taq polymerase. The amplified SAG1 gene was inserted into the Eco RI site of the plasmid pGEX-4T-3, and then it was expressed as a glutathione-S-transferase (GST) fusion protein in E. coli (DH 5 strain) according to the manufacture’s instructions (Amersham Pharmacia Biotech, USA). After centrifugation the bacteria were lyzed by a combination of detergent Triton X-100, lysozyme and ultrasonication. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | ultrasonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M urea solution containing 50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol (DTT) and 1 mM EDTA. |
| Refolding Buffer | 4 and then 2 M urea, 50 mM Tris-HCl (pH 8.0) with 1 mM DTT |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | DTT |
| Redox Agent Concentration | 1 mM |
| Refolding Protocol | The suspension was centrifuged and then the pellet dissolved in 8 M urea solution containing 50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol (DTT) and 1 mM EDTA. After 1 h incubation at room temperature and centrifugation, the supernatant was dialyzed against 4 and then 2 M urea solution at 4°C for 1 h each. Dialysis was done twice against a solution of 50 mM Tris-HCl (pH 8.0) with 1 mM DTT at 4°C and each lasting 1 h. This was followed by overnight dialysis at 4°C in the same buffer. The dialyzed sample was centrifuged and the supernatant recovered. The solution was purified by glutathione sepharose 4B according to manufacture’s instructions (Amersham Pharmacia Biotech, USA). The recovered supernatant was identified as purified rSAG1 and was used as antigen in ELISA. |
| Refolding Assay | Western Blot,SDS-PAGE,ELISA |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |