Expression of human mitochondrial thymidine kinase in escherichia coli: correlation between the enzymatic activity of pyrimidine nucleoside analogues and their inhibitory effect on bacterial growth
Section snippets
Bacterial strains and growth media
The bacterial strains used in this study were all derivatives of E. coli K12 and are listed in Table 1. As rich medium, Luria broth (LB) was used [8]. The minimal medium was AB medium [9] supplemented with 0.2% glucose, 0.2% vitamin-free casamino acids, and 1 μg/mL thiamine. When required, tryptophan was added at 50 μg/mL and uridine at 20 μg/mL (82 μM). Antibiotics were used in the following final concentrations: ampicillin, 100 μg/mL; tetracycline, 10 μg/mL; and kanamycin, 30 μg/mL.
Nucleoside analogues
Most
Activity of TK and dCK in crude extracts from the recombinant bacteria
To ascertain the expression of human TK2 in the engineered E. coli, crude extracts from strain SØ5338, containing the plasmid-borne coding sequence of human TK2 and the control strain SØ5292 (Table 1), were assayed for TK and dCK activity (Table 2). Both strains were devoid of endogenous E. coli TK activity due to the tdk-1 mutation. Since TK2 overlaps in its substrate specificity with dCK, the TK and dCK activities of strain SØ5218 (Table 1), harboring the coding sequence of human dCK on a
Discussion
5-Fluorouracil was designed for use as an antineoplastic agent [14], and 5FdU is one of its metabolites in vivo[15]. In cells, 5FdU has several metabolic routes. It is mainly phosphorylated by TK1 and TK2 to form 5FdU monophosphate, which can bind tightly to thymidylate synthase and inhibit the enzyme, thus impeding the transformation of dUMP to dTMP 15, 16. 5FdU can also be converted to 5-fluorouracil through a reversible reaction catalyzed by thymidine phosphorylase or, less efficiently,
Acknowledgements
This work was funded by the Swedish Medical Research Council, Swedish National Science Research Council, and grants from the EU Commission (BMH4-CT96-0479, to S.E.) and the Danish National Research Foundation (to J.N.).
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