Heterologous expression and characterization of the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of Toxoplasma gondii.
Heterologous expression and characterization of the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of Toxoplasma gondii.
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弓形虫双功能二氢叶酸还原酶-胸苷酸合酶的异源表达和表征。
DOI：
10.1021/bi952923q
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Santi,DV
中科院分区：
文献类型：
--
作者：
Trujillo,M;Donald,RG;Roos,DS;Greene,PJ;Santi,DV
We have expressed catalytically activeToxoplasma gondiidihydrofolate−thymidylate synthase (DHFR-TS) and the individual TS and DHFR domains inEscherichia coliusing the T7 promoter of pET-15b. DHFR-TS constituted approximately 10% of the total soluble cell protein and was purified using methotrexate-Sepharose chromatography to yield 10 mg of homogeneous DHFR-TS per liter of culture. The DHFR domain was recovered as insoluble inclusion bodies which could be unfolded and refolded to recover soluble, active enzyme. The TS domain was overexpressed as a soluble protein by growing the cells at 24 °C; this is the first report of the expression of an active TS domain from a bifunctional enzyme. ThekcatandKmvalues for DHFR-TS are similar to those of other previously characterized protozoan DHFRs and TSs. The antimicrobial antifolates, TMP and Pyr, inhibit DHFR activity of the bifunctional protein in accord with their effects in crude enzyme preparations andin vivosystems. Kinetic parameters andKivalues for TMP and Pyr with the isolated DHFR domain were identical to the values for DHFR in the bifunctional enzyme. Evidence of kinetic channeling of the dihydrofolate product of TS to the DHFR domain in the bifunctional enzyme was obtained by kinetic and inhibition studies. Properties such as yield, stability, and activities of the recombinantT. gondiiDHFR-TS provide clear advantages over other bifunctional DHFR-TSs as a model for future studies.
