CLONING AND EXPRESSION OF THE DIHYDROFOLATE REDUCTASE-THYMIDYLATE SYNTHASE GENE FROM TRYPANOSOMA-CRUZI

We have cloned, sequenced and expressed the Trypanosoma cruzi gene enc oding the bifunctional protein dihydrofolate reductase-thymidate synth ase (DHFR-TS). The strategy followed for the isolation of positive clo nes from a genomic library was based on the construction of a probe by the amplification of highly conserved sequences of the TS domain by t he 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 Esche richi coli, the entire coding sequence was amplified by polymerase cha in reaction and cloned into hte 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 st rain PA414. The gene is expressed as an active protein which constitut es approximately 2% of the total cell soluble protein. Recombinant bif unctional enzyme and the DHFR domain have been purified by methotrexat e-Sepharose chromatography to yield 1-2 mg of active DHFR-TS per litre of culture. Southern and electrophoretic analyses using the coding se quence as probe indicated that the T. cruzi enzyme is encoded by a sin gle 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.