Reche Gallardo, Pedro AntonioArrebola, ROlmo, ASanti, D VGonzález-Pacanowska, D.Ruiz Pérez, Luis Miguel2023-06-202023-06-2019940166-6851https://hdl.handle.net/20.500.14352/58262We 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.engjournal articlehttp://www.elsevier.com/wps/find/journaldescription.cws_home/506086/description#descriptionopen access612.017577.2Trypanosoma cruziDihydrofolate reductase-thymidylate synthaseProtozoal enzymeHeterologous expressionFolate metabolimsBioquímica (Biología)BiotecnologíaMicrobiología (Biología)Biología molecular (Biología)2302 Bioquímica3399 Otras Especialidades Tecnológicas2414 Microbiología2415 Biología Molecular