Purification of recombinant mAOX3 after expression in E. coli TP1000 cells
Total protein was quantified with the Bradford assay (Bradford, 1976). The activity was measured by monitoring the decrease in absorption at 600 nm in the presence of 250 μM benzaldehyde and 100 μM DCPIP. Specific enzyme activity is defined as the oxidation of 1 μM benzaldehyde per minute and milligram of enzyme under the assay conditions.
| Step | Volume | Total Protein | Benzaldehyde/DCPIP Activity | |||
|---|---|---|---|---|---|---|
| Total Activity | Specific Enzyme Activity | Yield | Fold Purification | |||
| ml | mg | IU | IU/mg | % | ||
| Crude extract | 280 | 17,513 | 163.3 | 0.009 | 77 | 0.8 |
| Cleared lysate | 280 | 18,354 | 212.9 | 0.012 | 100 | 1.0 |
| Flow-through | 280 | 18,597 | 170.8 | 0.009 | 80 | 0.8 |
| Ni-NTA | 30.4 | 35 | 3.6 | 0.104 | 1.71 | 9.0 |
| Chemical sulfuration | 49.4 | 32 | 5.0 | 0.158 | 2.34 | 13.6 |
| Superose 6 | 15.2 | 12 | 4.9 | 0.414 | 2.30 | 35.7 |