Refolding Record:
| Protein | |
|---|---|
| Protein Name | Recombinant grass pollen hybrid molecule |
| Abbreviated Name | Ph1 p6-Ph1 p2-Ph1 p5-Ph1 |
| SCOP Family | Group V grass pollen allergen |
| Structure Notes | |
| Organism | Phleum pratense (Common timothy) |
| UniProt Accession | MPAP6_PHLPR |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 548 |
| Molecular Weight | 59311.9 |
| Pi | 5.3 |
| Molecular Weight | 59311.9 |
| Disulphides | Unknown |
| Full Sequence |
GKATTEEQKLIEDVNASFRAAMATTANVPPADKYKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYNAAYNAADHAAPEDKYEAFVLHFSEALRIIAGTPEVHAVKPGAVPKVTFTVEKGSNEKHLAVLVKYEGDTMAEVELREHGSDEWVAMTKGEGGVWTFDSEEPLQGPFNFRFLTEKGMKNVFDDVVPEKYTIGATYAPEEIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKETTGGAYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQIPKVPPGPNITATYGDKWLDAKSTWYGKPTGAGPKDNGGACGYKDVDKPPFSGMTGCGNTPIFKSGRGCGSCFEIKCTKPEACSGEPVVVHITDDNEEPIAPYHFDLSGHAFGAMAKKGDEQKLRSAGELELQFRRVKCKYPEGTKVTFHVEKGSNPNYLALLVKYVNGDGDVVAVDIKEKGKDKWIELKESWGAIWRIDTPDKLTGPFTVRYTTEGGTKTEAEDVIPEGWKADTSYESK
|
| Notes | 4 Pollen allergens UniProtIDs are MPAP1_PHLPR, MPAP2_PHLPR, MPAP6_PHLPR, MPAT5_AMBTR |
| Expression | |
|---|---|
| Report | Linhart B, Hartl A, Jahn-Schmid B, Verdino P, Keller W, Krauth MT, Valent P, Horak F, Wiedermann U, Thalhamer J, Ebner C, Kraft D, Valenta R. (2005) J Allergy Clin Immunol, 115, 1010-1016 |
| Project Aim | Vaccine 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 | 5 h |
| Expression Vector | pET17p |
| Expression Protocol | Expression and purification of the hybrid molecule The hybrid molecule was expressed in Escherichia coli BL21 (DE3). Cells were grown in Luria Bertani-medium containing 100 μg/mL ampicillin to an OD600 of 0.8. Protein expression was induced by addition of isopropyl-β-thiogalactopyranoside to a final concentration of 0.5 mmol/L growing for 5 hours at 37°C. The hybrid was mainly detected in the inclusion body fraction. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.8 = 600 |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 mol/L urea, 100 mmol/L NaH2PO4, and 10 mmol/L TRIS, pH 8.0. |
| Refolding Buffer | 6 mol/L–1 mol/L urea gradient containing 500 mmol/L NaCl, 20% glycerol, and 20 mmol/L TRIS, pH 7.4 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 7.4 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 1.5 |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Inclusion bodies were solubilized in 8 mol/L urea, 100 mmol/L NaH2PO4, and 10 mmol/L TRIS, pH 8.0. The homogenate was centrifuged at 14,000g for 30 minutes, and the resulting supernatant was loaded on a Ni-NTA column (Quiagen, Hilden, Germany). The column-bound protein was refolded on the Ni-NTA column by using a linear 6 mol/L–1 mol/L urea gradient containing 500 mmol/L NaCl, 20% glycerol, and 20 mmol/L TRIS, pH 7.4, over a period of 1.5 hours.13 The soluble protein was eluted by addition of 250 mmol/L imidazole and dialyzed against H2O. The purified hybrid molecule remained soluble in water at a concentration of 1 mg/mL. |
| Refolding Assay | ELISA |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 20% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |