Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification

Detalhes bibliográficos
Autor(a) principal: Dias, Ana Margarida Gonçalves Carvalho
Data de Publicação: 2010
Tipo de documento: Dissertação
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10362/10924
Resumo: Dissertação apresentada para a obtenção do Grau de Mestre em Biotecnologia, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia
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spelling Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purificationDissertação apresentada para a obtenção do Grau de Mestre em Biotecnologia, pela Universidade Nova de Lisboa, Faculdade de Ciências e TecnologiaThe main objective of this work was the development of an affinity pair for the purification of recombinant proteins. In this work, ligands based on the Ugi Reaction and the 1,3,5-Triazine scaffold were synthesised in solid-phase and screened for binding to an affinity tag, an hexapeptide constituted by asparagine aminoacid (N). The ligands were tested against pure solutions of the hexapeptide and Green Fluorescence Protein (GFP), used as a model protein. The ligands that had the highest affinity for the hexapeptide and lowest affinity for the protein were selected for further studies with cellular extracts. The cellular extracts were produced in HEK 293T cells transfected with two designed vectors: one containing the GFP tagged with the affinity tag, and the other containing GFP without tag. The efficient expression of a recombinant GFP fused with the designed affinity tag was demonstrated. The cellular extracts were then loaded onto chromatographic columns containing the lead ligands immobilised onto agarose, and the amount of total protein and GFP bound and eluted noted. The results demonstrated that the Ugi ligands were less selective than the Triazine ligands for the hexapeptide. The triazine ligand 7,4 has been considered as the most selective for the designed affinity tag. In addition, preselected lead ligands for another hexapeptide (RW) of interest were studied. Mammalian cells HEK 293T were transfected with a vector expressing for GFP tagged with this peptide. The ligands immobilized onto agarose were loaded with cellular extracts, being noted that the lead A6C3 showed a high selectivity for the tag tested.Faculdade de Ciências e TecnologiaRoque, Ana CecíliaCoroadinha, AnaRUNDias, Ana Margarida Gonçalves Carvalho2014-01-02T17:08:01Z20102010-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/10924enginfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-05-22T17:14:45Zoai:run.unl.pt:10362/10924Portal AgregadorONGhttps://www.rcaap.pt/oai/openairemluisa.alvim@gmail.comopendoar:71602024-05-22T17:14:45Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification
title Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification
spellingShingle Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification
Dias, Ana Margarida Gonçalves Carvalho
title_short Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification
title_full Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification
title_fullStr Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification
title_full_unstemmed Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification
title_sort Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification
author Dias, Ana Margarida Gonçalves Carvalho
author_facet Dias, Ana Margarida Gonçalves Carvalho
author_role author
dc.contributor.none.fl_str_mv Roque, Ana Cecília
Coroadinha, Ana
RUN
dc.contributor.author.fl_str_mv Dias, Ana Margarida Gonçalves Carvalho
description Dissertação apresentada para a obtenção do Grau de Mestre em Biotecnologia, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia
publishDate 2010
dc.date.none.fl_str_mv 2010
2010-01-01T00:00:00Z
2014-01-02T17:08:01Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10362/10924
url http://hdl.handle.net/10362/10924
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Faculdade de Ciências e Tecnologia
publisher.none.fl_str_mv Faculdade de Ciências e Tecnologia
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron:RCAAP
instname_str Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron_str RCAAP
institution RCAAP
reponame_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
repository.mail.fl_str_mv mluisa.alvim@gmail.com
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