Table 4.
Characteristics of recombinant His-tagged NoxE of L. lactisTIL46 and the different variants produced in E. coli after purification on Ni-NTA agarose resin
| Protein | Amta (mg) | % Purityb | Particles with expected sizec | Sp actd (U/mg) | FAD/NoxE ratioe |
|---|---|---|---|---|---|
| NoxE | 2.5 | 100 | + | 130 ± 9 | 1 |
| NoxE_A144T | 2.6 | 100 | + | 137 ± 10 | − |
| NoxE_K384N | 3.5 | 100 | + | 139 ± 11 | − |
| NoxE_A303T | 0.3 | 9 | − | 0.13 ± 0.1 | ND |
| NoxE_A303G | 0.3 | 20 | − | 9 ± 8 | ND |
| NoxE_L299T | 6 | 100 | + | 35 ± 3 | 0.9 |
| NoxE_A300T | 2 | 100 | + | 123 ± 12 | 1 |
| NoxE_N302S | 0.4 | 38 | − | 53 ± 10 | ND |
| NoxE_G307S | 0.25 | 9 | − | 1 ± 1 | ND |
| NoxE_G307A | 0.35 | 6 | − | 2 ± 1 | ND |
Amount of protein retrieved in the elution fraction from the Ni-NTA column.
Estimated by SDS-PAGE (see Fig. S2 in the supplemental material).
Presence (+) or absence (−) of particles with a hydrodynamic diameter of 8.7 nm (corresponding to an apparent molecular mass of 100 kDa) as determined by dynamic light scattering (see Fig. S3 in the supplemental material).
The data are means of 2 determinations ± standard deviations and take into account the percent purity of protein preparations estimated by SDS-PAGE. Activity was determined after reactivation with cysteine and FAD.
−, not determined; ND, not detected.