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...

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Detalles Bibliográficos
Autores: Reche Gallardo, Pedro Antonio, Arrebola, R, Olmo, A, Santi, D V, González-Pacanowska, D., Ruiz Pérez, Luis Miguel
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
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oai_identifier_str oai:docta.ucm.es:20.500.14352/58262
network_acronym_str ES
network_name_str España
repository_id_str
spelling Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruziReche Gallardo, Pedro AntonioArrebola, ROlmo, ASanti, D VGonzález-Pacanowska, D.Ruiz Pérez, Luis Miguel612.017577.2Trypanosoma cruziDihydrofolate reductase-thymidylate synthaseProtozoal enzymeHeterologous expressionFolate metabolimsBioquímica (Biología)BiotecnologíaMicrobiología (Biología)Biología molecular (Biología)2302 Bioquímica3399 Otras Especialidades Tecnológicas2414 Microbiología2415 Biología MolecularWe 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.ElsevierUniversidad Complutense de Madrid19941994-01-0119941994-01-01journal articlehttp://purl.org/coar/resource_type/c_6501info:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/20.500.14352/58262reponame:Docta Complutenseinstname:Universidad Complutense de Madrid (UCM)Inglésengopen accesshttp://purl.org/coar/access_right/c_abf2info:eu-repo/semantics/openAccessoai:docta.ucm.es:20.500.14352/582622026-06-02T12:44:21Z
dc.title.none.fl_str_mv Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruzi
title Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruzi
spellingShingle Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruzi
Reche Gallardo, Pedro Antonio
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
title_short Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruzi
title_full Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruzi
title_fullStr Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruzi
title_full_unstemmed Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruzi
title_sort Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruzi
dc.creator.none.fl_str_mv Reche Gallardo, Pedro Antonio
Arrebola, R
Olmo, A
Santi, D V
González-Pacanowska, D.
Ruiz Pérez, Luis Miguel
author Reche Gallardo, Pedro Antonio
author_facet Reche Gallardo, Pedro Antonio
Arrebola, R
Olmo, A
Santi, D V
González-Pacanowska, D.
Ruiz Pérez, Luis Miguel
author_role author
author2 Arrebola, R
Olmo, A
Santi, D V
González-Pacanowska, D.
Ruiz Pérez, Luis Miguel
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Universidad Complutense de Madrid
dc.subject.none.fl_str_mv 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
topic 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
description 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.
publishDate 1994
dc.date.none.fl_str_mv 1994
1994-01-01
1994
1994-01-01
dc.type.none.fl_str_mv journal article
http://purl.org/coar/resource_type/c_6501
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://hdl.handle.net/20.500.14352/58262
url https://hdl.handle.net/20.500.14352/58262
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:Docta Complutense
instname:Universidad Complutense de Madrid (UCM)
instname_str Universidad Complutense de Madrid (UCM)
reponame_str Docta Complutense
collection Docta Complutense
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,301629

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