TABLE 1.
Total yield and kinetic parameters of purified TrxR induced at different expression levels in LB medium with the pBAD expression system
| Arabinose (%) | pSUABC | Recovereda (mg/liter) | Recovereda (mg/liter/OD600) | kcat (min−1) | Km (mM) | kcat/Km (min−1 mM−1) | U/mgb | Recovered selenoproteinc (mg/liter/OD600) |
|---|---|---|---|---|---|---|---|---|
| 0.0002 | − | 5.8 | 3.8 | 25 | 0.15 | 167 | 0.2 | 0.02 |
| + | 9.3 | 5.7 | 159 | 0.13 | 1,223 | 1.4 | 0.23 | |
| 0.005 | − | 13.4 | 9.5 | 123 | 0.18 | 683 | 1.1 | 0.30 |
| + | 29.2 | 18.2 | 315 | 0.13 | 2,423 | 2.7 | 1.40 | |
| 0.2 | − | 13.6 | 9.8 | 81 | 0.12 | 675 | 0.7 | 0.20 |
| + | 24.2 | 15.2 | 646 | 0.17 | 3,800 | 5.6 | 2.43 |
a
Total recombinant TrxR (mixture of truncated and full-length forms) recovered in purification, given as yield per liter of original medium or per liter and OD600 unit.
b
One unit is defined as the reduction of 1 μmol of DTNB per min in the standard DTNB assay (2). The specific activity of native mammalian TrxR is approximately 35 U/mg (2).
c
Selenoprotein (full-length TrxR) recovered in the purification, given as yield per original culture volume and OD600 unit, as calculated from units per milligram and total recovered yield, assuming 35 U/mg for the full- length selenoprotein (2).