Selective Fractionation of Human Flavin-Containing Monooxygenase Maltose Binding Protein
| A Fraction | Protein (mg) | 5-DPT N-Oxygenation Activity1-a (nmol/min/mg of protein) |
|---|---|---|
| Lys158 FMO3 MBP | ||
| Bacterial lysate | 44.31-b | 0.19 ± 0.1 |
| Amylose resin fraction | 13.21-c | 6.89 ± 0.5 |
| PEG 8000 7% pellet | 1.7 | 5.22 ± 0.4 |
| PEG 8000 14% pellet | 5.5 | 13.02 ± 0.3 |
| PEG 8000 14% supernatant | 5.8 | 0.24 ± 0.2 |
| B | ||
| Glu158 FMO3 MBP | ||
| Bacterial lysate | 47.21-b | 0.02 ± 0 |
| Amylose resin fractions | 9.11-c | 4.94 ± 0.4 |
| 7% PEG 8000 pellet | 2.0 | 3.18 ± 0 |
| 14% PEG 8000 pellet | 1.6 | 26.46 ± 0.8 |
| 14% PEG 8000 supernatant | 4.0 | 0.26 ± 0.1 |
↵1-a 5-DPT N-oxygenase activity was determined by the HPLC method described in Materials and Methods with 3–5 determinations ± SD. Fractionation of the protein on SDS PAGE is shown in fig. 1A. Mock transformations of E. coli without the cDNA encoding the FMO3-MBP failed to produce the 100 kDA protein or any 5-DPTN-oxygenase activity. The limit of detection was approximately 5 pmol 5-DPTNO/min/mg of protein.
↵1-b A typical 4-liter preparation contained 45–55 mg of total protein with the specific activity listed. This protein was placed on the amylose affinity column resin.
↵1-c A portion of the eluant from the amylose affinity column (i.e. 20–30%) was used for subsequent fractionation.