Refolding Record:
| Protein | |
|---|---|
| Protein Name | Non-specific lipid-transfer protein |
| Abbreviated Name | nsLTP1 |
| SCOP Family | Plant lipid-transfer and hydrophobic proteins |
| Structure Notes | |
| Organism | Triticum aestivum (Wheat) |
| UniProt Accession | A0MAU4 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 185 |
| Molecular Weight | 18820.8 |
| Pi | 8.43 |
| Molecular Weight | 18820.8 |
| Disulphides | Unknown |
| Full Sequence |
MAARRSGAVVAALMALLVGLAGADFAADRAECADKLMGLATCLTYVQLAATARSPTPDCCSGFRQVLGVSKKCLCVLVKDRDEPTLGIKFNVTRAMNLPSACNIPATFSDCPKILNMSPDSKEAEIFKQYGIEHEGKNATAGGSAAVTGTSGGKSADAAAGAGRHTAVVFAVVVSALLASVLVLA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lullien-Pellerin V, Devaux C, Ihorai T, Marion D, Pahin V, Joudrier P, Gautier MF. (1999) Eur J Biochem., 260, 861-868 |
| Project Aim | Undefined |
| 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 | 3 h |
| Expression Vector | pLysS |
| Expression Protocol | E. coli BL21(DE3)pLysS containing a recombinant expression vector was grown overnight at 37 °C in Luria–Bertani medium with 0.1 mg·mL−1 ampicillin and 0.025 mg·mL−1 chloramphenicol. Cells were diluted 1 : 60, allowed to grow until absorbance at 600 nm reached 0.5 and then induced by the addition of isopropyl-β- d-thiogalactopyranoside to a final concentration of 0.1 m m in the culture medium. After 3 h of induction, bacteria were harvested by centrifugation and stored at –20 °C. For recombinant protein purification, cells were resuspended in 1 : 20 of the culture volume of lysis buffer (50 m m Tris/HCl, pH 7.8, 1 m m EDTA) and broken, after two freeze-thaw cycles, by passing four times through a French Press at 35 MPa. The resulting lysate was incubated 30 min at 37 °C with 12 U·mL−1 Benzon Nuclease to digest the nucleic acids; the bacterial lysate was centrifuged for 10 min at 13 500 g. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 600 |
| Cell Disruption Method | Freeze/Thaw+French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | buffer A (50 m m Tris/HCl, pH 7.8, 0.1 m KCl, 10 m m EDTA) |
| Solubilization Buffer | buffer A containing 2% 2-mercaptoethanol and 7.5 m urea |
| Refolding Buffer | 10 vol. of buffer A |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.8 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 1 mg·mL−1 |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The pellet, containing the inclusion bodies, was resuspended in three lysate volumes of washing buffer (50 m m Tris/HCl, pH 7.8, 0.1 m KCl, 10 m m EDTA), kept at room temperature for 10 min, and centrifuged for 5 min as above. The washing step was repeated twice with buffer A (50 m m Tris/HCl, pH 7.8, 0.1 m KCl, 1 m m EDTA) and the pellet was resuspended, with constant stirring, in buffer A containing 2% 2-mercaptoethanol and 7.5 m urea, at a protein concentration of 1 mg·mL−1. After 1 h, particulate material was removed by centrifugation as described before and one volume of the supernatant was diluted with 10 vol. of buffer A and stirred for 1 h. Denaturing agents were then removed by dialysis against buffer A for 12 h and water for 24 h using Spectrapor dialysis tubing (Spectrum, 3000 Mr cut-off). After dialysis, samples were centrifuged at 12 000 g for 2 h, concentrated by ultrafiltration using a Microsep 3K (Filtron ) and lyophilized. |
| Refolding Assay | Fluorescence,Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |