Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruzi
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...
| Autores: | , , , , , |
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| Tipo de recurso: | artículo |
| Fecha de publicación: | 1994 |
| País: | España |
| Institución: | Universidad Complutense de Madrid (UCM) |
| Repositorio: | Docta Complutense |
| Idioma: | inglés |
| OAI Identifier: | oai:docta.ucm.es:20.500.14352/58262 |
| Acceso en línea: | https://hdl.handle.net/20.500.14352/58262 |
| Access Level: | acceso abierto |
| Palabra clave: | 612.017 577.2 Trypanosoma cruzi Dihydrofolate reductase-thymidylate synthase Protozoal enzyme Heterologous expression Folate metabolims Bioquímica (Biología) Biotecnología Microbiología (Biología) Biología molecular (Biología) 2302 Bioquímica 3399 Otras Especialidades Tecnológicas 2414 Microbiología 2415 Biología Molecular |
| Sumario: | 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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