Table 1. Screening for recombinant T. brucei DHFR and TS activities as expressed in E. coli.
| Construct | N-terminal tag | E. coli strain | Specific activity, mU mg-1 | Ratio DHFR:TS | |
|---|---|---|---|---|---|
| DHFR | TS | ||||
| Tsf blank vector | N/A | thyA- a | 2.1 ± 0.2 | Not detected | — |
| Tsf blank vector | N/A | thyA+ | 3.6 ± 0.2 | Not detected b | — |
| TbDHFR | His6 | thyA+ | 2000 ± 76 | Not detected b | — |
| TbDHFR-TS | His6 | thyA- | 210 ± 14 | Not detected | — |
| Tsf-TbDHFR-TS | Tsf | thyA- | 1200 ± 7 | 8.8 ± 0.1 | 137:1 |
Enzyme activity was determined in clarified lysates using the spectrophotometric assays described in the methods. Values are the means and standard deviations of triplicate assays from a single experiment.
a) Grown in presence of 50 μg ml-1 thymine to compensate for lack of endogenous TS
b) The spectrophotometric assay is insufficiently sensitive to detect endogenous TS activity in the thyA+ E. coli host