Biblioteca de la Universidad Complutense de Madrid

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

Impacto



Reche, Pedro A y Arrebola, R y Olmo, A y Santi, D V y González-Pacanowska, D. y 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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URL Oficial: http://www.elsevier.com/wps/find/journaldescription.cws_home/506086/description#description



Resumen

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.


Tipo de documento:Artículo
Palabras clave:Trypanosoma cruzi; Dihydrofolate reductase-thymidylate synthase; Protozoal enzyme; Heterologous expression; Folate metabolims
Materias:Ciencias Biomédicas > Biología > Bioquímica
Ciencias Biomédicas > Biología > Biotecnología
Ciencias Biomédicas > Biología > Microbiología
Ciencias Biomédicas > Biología > Biología molecular
Código ID:9355
Depositado:17 Aug 2009 08:58
Última Modificación:06 Oct 2014 12:01

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