Refolding Record:
| Protein | |
|---|---|
| Protein Name | Brazzein |
| Abbreviated Name | Brazzein |
| SCOP Family | Plant defensins |
| Structure Notes | |
| Organism | Pentadiplandra brazzeana |
| UniProt Accession | P56552 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 203 |
| Molecular Weight | 23063.2 |
| Pi | 8.9295 |
| Molecular Weight | 23063.2 |
| Disulphides | 4 |
| Full Sequence |
ATSTKKLH KEPATLIKAI DGDTVKLAYK GQPATFRLLL
VDTPETKHPK KGVEKYGPEA SAFTKKAVEN AKKIEVEFDK GQRTDKYGRG LAYIYADGKA VNEALVRQGL AKVAYVYKPN NTHEQHLRKS EAQAKKEKLN IWSEDNADSG H MDKCKKVYEN YPVSKCQLAN QCNYDCKLDK HARSGECFYD EKRNLQCICD
YCEY
|
| Notes | Fusion partner: staphylococcal nuclease UniProt: NUC_STAAU, mature protein with Met residues mutated to Ala Brazzein sequence lacks N-terminal Glu residue |
| Expression | |
|---|---|
| Report | Assadi-Porter FM, Aceti DJ, Cheng H, Markley JL (2000) Archives Biochemistry and Biophysics, 376, 252-258 |
| Project Aim | Recombinant Protein Expression |
| Fusion | N-terminal staphylococcal nuclease |
| 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 | 3h |
| Expression Vector | pET3a |
| Expression Protocol | 1L LB medium containing 34microg/ml chloramphenicol and 100microg/ml ampicillin was inoculated with 5ml overnight culture. The cells were grown at 37degC until A600 reached 0.8-1.0, at which time expression was induced with 0.1mM IPTG. Cells were grown a further 3h, then harvested and stored at -70degC. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.8-1.0 |
| Cell Disruption Method | Freeze/Thaw+French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50mM TrisHCl pH 8.0, 2mM EDTA |
| Solubilization Buffer | 6M GdmCl, 10mM EDTA, 100mM DTT |
| Refolding Buffer | acetic acid pH 3.8-4.0 |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | yes |
| Refolding pH | 3.9 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 36-48h |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Cells were freeze/thawed once, then 4-5g cells was resuspended in 50ml of 50mM TrisHCl pH 8.0, 2mM EDTA, 10mM PMSF. The cells were treated with 10mM CaCl2 for 15min and passed through a French press three times. The cells were then centrifuged (15min, 12000g). The cell pellet was washed three times with 9 volumes of wash buffer, the second wash also containing 0.5% (v/v) Triton X-100 and 100mM NaCl. In each wash, the slurry was stirred gently for 5min and then centrifuged (12000g, 10min, 4degC). The final pellet was resuspended in 50ml solubilization buffer and stirred for 2-3h at room temperature. The solution was the dialyzed overnight at 4degC against 4L deionized water containing 3.5ml acetic acid (pH 3.8-4.0). The precipitate was removed by centrifugation (12000g). The supernatant was diazlyed three more times against dH2O and acetic acid for 36-48h. For oxidation of sulfhydryl groups, the dialysate was then diluted with 4-5 volumes of 200mM Tris-acetic acid pH 8.0 to a final concentration of 0.3-0.7mg/ml and this solution was stirred at room temperature for 24h. The solution was then concentrated to a final volume of 20-50ml, then dialyzed against 10L of 0.3M NaCl and twice against 10L of dH2O, the protein was then lyophilized. |
| Refolding Assay | 1H chemical shift (ppm),SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 60-70% |
| Purity | 80% |
| Notes | n/a |