RT Journal Article
T1 Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruzi
A1 Reche Gallardo, Pedro Antonio
A1 Arrebola, R
A1 Olmo, A
A1 Santi, D V
A1 González-Pacanowska, D.
A1 Ruiz Pérez, Luis Miguel
AB 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.
PB Elsevier
SN 0166-6851
YR 1994
FD 1994
LK https://hdl.handle.net/20.500.14352/58262
UL https://hdl.handle.net/20.500.14352/58262
LA eng
DS Docta Complutense
RD 8 abr 2025