Refolding Record:
| Protein | |
|---|---|
| Protein Name | Prion |
| Abbreviated Name | Prion |
| SCOP Family | Prion-like |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | P04925 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 209 |
| Molecular Weight | 22932.2 |
| Pi | 9.56 |
| Molecular Weight | 22932.2 |
| Disulphides | Unknown |
| Full Sequence |
KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGTWGQPHGGGWGQPHGGSWGQPHGGSWGQPHGGGWGQGGGTHNQWNKPSKPKTNLKHVAGAAAAGAVVGGLGGYMLGSAMSRPMIHFGNDWEDRYYRENMYRYPNQVYYRPVDQYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCVTQYQKESQAYYDGRRS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Inanami O, Hashida S, Iizuka D, Horiuchi M, Hiraoka W, Shimoyama Y, Nakamura H, Inagaki F, Kuwabara M. (2005) Biochemical and Biophysical Research Com, 335, 785-792 |
| Project Aim | Structural Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 6 h |
| Expression Vector | pRSETb |
| Expression Protocol | Expression and purification of recombinant moPrP mutants. The expression plasmids were introduced into E. coli BL21(DE3)LysS. Protein expression was induced by adding IPTG to a final concentration at 0.5 mM. Four to six hours after induction, bacterial cells were collected and inclusion bodies were prepared as described elsewhere [24]. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M GdnHCl in 20 mM phosphate buffer |
| Refolding Buffer | 10 mM acetate buffer (pH 4.0) |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 4.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | 48 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The recombinant moPrP was further purified by Ni2+-immobilized metal affinity chromatography using Ni2+-charged chelating Sepharose (Qiagen) and a stepwise elution gradient from pH 6.5 to 4.3 in the presence of 8 M urea. After dialysis against 10 mM acetate buffer (pH 4.0) for 48 h, recombinant moPrP containing an intramolecular disulfide bond was purified by reverse-phase HPLC using TSKgel phenyl-5PW RP and a 30–50% linear gradient of acetonitrile with 0.05% trifluoroacetic acid. The purified recombinant moPrP was dialyzed against 10 mM acetate buffer (pH 4.0) and stored at −20 °C until use. The protein concentration was determined by measuring the UV absorption at 276 nm using an extinction coefficient of 39,425 cm2 M−1. Protein purity was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), followed by Commasie brilliant blue staining. All mutants were at least 95% pure as judged by the SDS–PAGE. |
| Refolding Assay | Circular Dichroism (uv-CD) |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 95% |
| Purity | n/a |
| Notes | n/a |