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Título:
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Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruzi
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Autores:
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Reche, Pedro A ;
Arrebola, R ;
Olmo, A ;
Santi, D V ;
González-Pacanowska, D. ;
Ruiz Pérez, Luis Miguel
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Tipo de documento:
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texto impreso
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Editorial:
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Elsevier, 1994
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Dimensiones:
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application/pdf
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Nota general:
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info:eu-repo/semantics/openAccess
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Idiomas:
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Palabras clave:
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Estado = Publicado
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Materia = Ciencias Biomédicas: Biología: Bioquímica
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Materia = Ciencias Biomédicas: Biología: Biotecnología
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Materia = Ciencias Biomédicas: Biología: Microbiología
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Materia = Ciencias Biomédicas: Biología: Biología molecular
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Tipo = Artículo
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Resumen:
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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.
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En línea:
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https://eprints.ucm.es/id/eprint/9355/1/02.Reche_etal_JMP_1994.pdf
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