Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells

Detalhes bibliográficos
Autor(a) principal: Cunha, Bárbara Martins Paiva da
Data de Publicação: 2011
Tipo de documento: Dissertação
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10362/6659
Resumo: Dissertation to obtain a Master Degree in Biotechnology
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spelling Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cellsCarboxylesterase 2ManufacturingSecretionMammalian cellsScale-upDissertation to obtain a Master Degree in BiotechnologyThe carboxylesterase 2 enzyme (CES2), the main carboxylesterase (CES) expressed in human intestine, has a key role in the metabolism of ester containing xenobiotics including the activation of several prodrugs and therefore, it can influence significantly the bioavailability of these compounds. CES2 is receiving an increasing attention due to its potential application in anti-cancer combined therapies, for the treatment of different pathologies like colon adenocarcinoma and malignant glioma. Unlike carboxylesterase 1 (CES1), the structure of this enzyme is not yet known. Since the knowledge of the structure of this protein will be crucial for the understanding of its properties, it becomes relevant the establishment of a process for the production and purification at the milligram level. The development and optimization of human recombinant CES2 production process, using human embryonic kidney cells (HEK-293T cells), is described in this thesis. First, the C-terminal 10xHistidine tag CES2 (CES2-10xHis) was produced using adherent cultures, and then the process was transferred to suspension cultures with serum-free media and finally scaled up to a five-litre stirred tank bioreactor. An affinity chromatography purification process was applied to obtain the CES2-10xHis protein, with a high purity grade. Despite being an Endoplasmic Reticulum (ER) anchored protein, secretion of CES2 to serum free media was achieved due to the presence of the in frame C-terminal 10xHistidine tag. With this elegant procedure we avoided the addition of extra N-terminal signaling sequences or the mutation or deletion of the C-terminal Histidine - Threonine - Glutamic acid - Leucine (HTEL) motif responsible for anchoring the protein in the lumen of the ER. Secretion contributed to a good production yield; however, the global process yield was quite low, due to difficulties in the purification step, which we attributed to protein aggregation.Faculdade de Ciências e TecnologiaSimplício, AnaCoroadinha, AnaRUNCunha, Bárbara Martins Paiva da2012-01-10T16:22:20Z20112011-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/6659enginfo: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:RCAAP2024-03-11T03:37:50Zoai:run.unl.pt:10362/6659Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:16:55.487265Repositório 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 Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells
title Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells
spellingShingle Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells
Cunha, Bárbara Martins Paiva da
Carboxylesterase 2
Manufacturing
Secretion
Mammalian cells
Scale-up
title_short Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells
title_full Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells
title_fullStr Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells
title_full_unstemmed Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells
title_sort Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells
author Cunha, Bárbara Martins Paiva da
author_facet Cunha, Bárbara Martins Paiva da
author_role author
dc.contributor.none.fl_str_mv Simplício, Ana
Coroadinha, Ana
RUN
dc.contributor.author.fl_str_mv Cunha, Bárbara Martins Paiva da
dc.subject.por.fl_str_mv Carboxylesterase 2
Manufacturing
Secretion
Mammalian cells
Scale-up
topic Carboxylesterase 2
Manufacturing
Secretion
Mammalian cells
Scale-up
description Dissertation to obtain a Master Degree in Biotechnology
publishDate 2011
dc.date.none.fl_str_mv 2011
2011-01-01T00:00:00Z
2012-01-10T16:22:20Z
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/10362/6659
url http://hdl.handle.net/10362/6659
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
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