Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruzi



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Reche, Pedro A and Arrebola, R and Olmo, A and Santi, D V and González-Pacanowska, D. and Ruiz Pérez, Luis Miguel (1994) Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruzi. Molecular and Biochemical Parasitology, 65 (2). pp. 247-58. ISSN 0166-6851

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We have cloned, sequenced and expressed the Trypanosoma cruzi gene encoding the bifunctional protein dihydrofolate reductase-thymidylate synthase (DHFR-TS). The strategy followed for the isolation of positive clones from a genomic library was based on the construction of a probe by the amplification of highly conserved sequences of the TS domain by the polymerase chain reaction. Translation of the open reading frame of 1563 bp yields a polypeptide of 521 amino acids with a molecular mass of 58829 Da. For heterologous expression of T. cruzi DHFR-TS in Escherichia coli, the entire coding sequence was amplified by polymerase chain reaction and cloned into the plasmid vector pKK223.3. The presence of catalytically active DHFR-TS was demonstrated by complementation of the Thy- E. coli strain chi 2913 and the DHFR- Thy- E. coli strain PA414. The gene is expressed as an active protein which constitutes approximately 2% of the total cell soluble protein. Recombinant bifunctional enzyme and the DHFR domain have been purified by methotrexate-Sepharose chromatography to yield 1-2 mg of active DHFR-TS per litre of culture. Southern and electrophoretic analyses using the coding sequence as probe indicated that the T. cruzi enzyme is encoded by a single copy gene which maps to two bands of approximately 990 kb and 1047 kb. It appears that T. cruzi is diploid for the DHFR-TS gene which is located on two different-sized homologous chromosomes.

Item Type:Article
Uncontrolled Keywords:Trypanosoma cruzi; Dihydrofolate reductase-thymidylate synthase; Protozoal enzyme; Heterologous expression; Folate metabolims
Subjects:Medical sciences > Biology > Biochemistry
Medical sciences > Biology > Biotechnology
Medical sciences > Biology > Microbiology
Medical sciences > Biology > Molecular biology
ID Code:9355
Deposited On:17 Aug 2009 08:58
Last Modified:13 Dec 2018 12:48

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