Refolding Record:
| Protein | |
|---|---|
| Protein Name | 30K protein |
| Abbreviated Name | n/a |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Bombyx mori |
| UniProt Accession | Q17185 |
| 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 | 256 |
| Molecular Weight | 29666.8 |
| Pi | 6.24 |
| Molecular Weight | 29666.8 |
| Disulphides | Unknown |
| Full Sequence |
MRLTLFAFVLAVCALASNATLAPRTDDVLAEQLYMSVVIGEYETAIAKCSEYLKEKKGEVIKEAVKRLIENGKRNTMDFAYQLWTKDGKEIVKSYFPIQFRVIFTEQTVKLINKRDHHALKLIDQQNHNKIAFGGSKDKTSKKVSWKFTPVLENNRVYFKIMSTEDKQYLKLDNTKGSSDDRIIYGDSTADTFKHQWYLEPSMYESDVMFFVYNREYNSVMTLDEDMAANEDREALGHSGEVSGYPQLFAWYIVPY
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kim EJ, Park HJ, Park TH. (2003) Biochemical and Biophysical Research Com, 308, 523-528 |
| Project Aim | Drug Studies |
| 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)pLysE |
| Expression Temp | 0.0 |
| Expression Time | 4 h |
| Expression Vector | pET22b |
| Expression Protocol | Protein expression, purification, and refolding. The pET-22b(+) carrying 30Kc6 was introduced into E. coli strain BL21(DE3) and BL21(DE3)pLysE. The transformed bacteria were grown to OD600 of 0.5, induced with 0.5 mM isopropyl-β-thiogalactopyranoside (IPTG), and then incubated for 4 h. The cells were harvested by centrifugation and resuspended in 4 ml of lysis buffer (10 mM Tris–HCl, 150 mM NaCl, and 1 mM EDTA, pH 8.0) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) for each 100 ml of culture. Lysozyme (0.5 mg/ml) was added and the mixture was incubated on ice for 30 min. The suspended cells were disrupted by sonication (Vibracell, 4 × 15 s) and centrifuged at 4 °C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Ni-NTA agrose chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M guanidine hydrochloride |
| Refolding Buffer | linear urea reverse gradient (6–0 M) |
| Pre-Refolding Purification | Ni-NTA agrose chromatography |
| Tag Cleaved | no |
| Refolding pH | 0.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The precipitate containing inclusion bodies was solubilized in 6 M guanidine hydrochloride overnight at 4 °C. This solution was loaded on a HisTrap column (Amersham Bioscience) and the column was washed with buffer containing 6 M urea and 20 mM imidazole several times to remove the nonspecific binding. Refolding of the bound protein was performed in an FPLC (Bio-Rad, Biologic HR) using a linear urea reverse gradient (6–0 M). The total volume and flow rate of the buffer used in the linear gradient were 30 ml and 0.5 ml/min, respectively. Finally, the refolded protein was eluted with elution buffer containing 500 mM imidazole. The eluted 30K protein was desalted into the distilled water to remove the imidazole using a HiTrap desalting column (Amersham Bioscience) and concentrated using a lyophilizer. |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |