Refolding Record:
| Protein | |
|---|---|
| Protein Name | Antimicrobial peptide 1 |
| Abbreviated Name | MIAMP1 |
| SCOP Family | Plant antimicrobial protein MIAMP1 |
| Structure Notes | |
| Organism | Macadamia nut (Macadamia integrifolia) |
| UniProt Accession | P80915 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 78 |
| Molecular Weight | 8138.0 |
| Pi | 9.07 |
| Molecular Weight | 8138.0 |
| Disulphides | 3 |
| Full Sequence |
SAFTVWSGPGCNNRAERYSKCGCSAIHQKGGYDFSYTGQTAALYNQAGCSGVAHTRFGSSARACNPFGWKSIFIQC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Harrison SJ, McManus AM, Marcus JP, Goulter KC, Green JL, Nielsen KJ, Craik DJ, Maclean DJ, Manners JM. (1999) Protein Expression and Purification, 15, 171-177 |
| Project Aim | Purification & characterization |
| Fusion | N-terminal methionine |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 00 |
| Expression Vector | pET17b |
| Expression Protocol | Cultures for protein expression were grown in ZCYM (10) and supplemented with 100 ug/mL ampicillin and 50 ug/mL kanamycin. Cultures were incubated at 37°C until OD600 reached 0.6 whereupon protein expression was induced by the addition of 400 uM IPTG. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 600 |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M guanidine hydrochloride |
| Refolding Buffer | 50 mM Tris-buffered saline containing 1.5 M urea |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no |
| Refolding pH | 0.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | 6 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Following growth and induction of the transformed E. coli, cells were centrifuged for 10 min at 5000 rpm and resuspended in 20 mM Tris (pH 9) containing 10 mM EDTA and 0.1 mM PMSF. The cells were lysed using a French Pressure Cell (Sim Aminco) at 1000 psi setting in a 40-mL pressure cell. Lysed cells were then incubated for 2 h at 4°C in the presence of DNase. Following incubation the mixture was centrifuged at 15,000 rpm for 30 min to separate the insoluble material from the soluble fraction. The supernatant was passed over an anion-exchange column (Q Sepharose Fast Flow, Pharmacia) equilibrated with 20 mM Tris, pH 9. The flowthrough from this column represented the basic (pI > 9) protein fraction. The pellet of insoluble material from the initial centrifugation was resolubilized overnight at 4°C with gentle shaking using 6 M guanidine hydrochloride and then centrifuged at 15,000 rpm to remove the remaining insoluble material after resolublization. This solution was then rapidly diluted (1:20) with 50 mM Tris-buffered saline containing 1.5 M urea and shaken gently for 6 h. The urea and guanidine were removed by dialysis against 10 mM 2-(morpholino)ethanesulfonic acid (MES) 2 pH 6 plus 10 mM EDTA. After the completion the sample was removed from dialysis and frozen. |
| Refolding Assay | HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |