Nature-based Peptide and Protein Scaffolds as Enzyme Mimetics
Autor(a) principal: | |
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Data de Publicação: | 2018 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10362/52950 |
Resumo: | Protein and peptide scaffolds are of interest for many bioengineering applications. The incorporation of catalytic activity in such structures is of major importance, although it represents a challenge. Several strategies are being used to obtain peptide catalysts and protein/peptide-based hydrogels, but the merging of the two fields is still in infancy. This work aims to study a new peptide scaffold, based on the small domain peptide, which is part of PTEN, a tumor suppressor protein. To fully characterize this peptide scaffold, the peptide was produced by two strategies; (i) small domain fused with GFP (SD-GFP) and; (ii) the small domain without fusion partner (SD). Both strategies were developed by using bacterial cells as hosts and purified by chromatographic-based techniques. The soluble SD-GFP scaffold was obtained with 93% purity, and showed catalytic activity towards pNPP, presenting a rate of 5x10-4 s-1, which is an order of magnitude lower than the kcat of PTEN towards pNPP. The production of SD-GFP also led to the formation of inclusion bodies, which were solubilized and matrix-assisted refolded on-column with 69% purity, leading to a self-supporting hydrogel at 4 ºC. The gel was washed by using three cycles of PBS and distilled water, which allowed to increase hydrogel purity. This corroborates the fact that hydrogel network is being formed by SD-GFP. The SD peptide was produced in the soluble form in bacterial cells and was purified yielding 90% of purity. The presence of paired cysteines in the SD peptide sequence lead to the formation of disulfide bridges forming tetrameric assemblies. These assemblies present an α-helical structure but are not catalytically active. In the future, the peptide should be in its reduced form (in monomer), so that cysteines can act as a nucleophile, catalyzing the dephosphorylation of pNPP. |
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Nature-based Peptide and Protein Scaffolds as Enzyme MimeticscatalysispeptidePTENscaffoldself-assemblyDomínio/Área Científica::Engenharia e Tecnologia::Engenharia QuímicaProtein and peptide scaffolds are of interest for many bioengineering applications. The incorporation of catalytic activity in such structures is of major importance, although it represents a challenge. Several strategies are being used to obtain peptide catalysts and protein/peptide-based hydrogels, but the merging of the two fields is still in infancy. This work aims to study a new peptide scaffold, based on the small domain peptide, which is part of PTEN, a tumor suppressor protein. To fully characterize this peptide scaffold, the peptide was produced by two strategies; (i) small domain fused with GFP (SD-GFP) and; (ii) the small domain without fusion partner (SD). Both strategies were developed by using bacterial cells as hosts and purified by chromatographic-based techniques. The soluble SD-GFP scaffold was obtained with 93% purity, and showed catalytic activity towards pNPP, presenting a rate of 5x10-4 s-1, which is an order of magnitude lower than the kcat of PTEN towards pNPP. The production of SD-GFP also led to the formation of inclusion bodies, which were solubilized and matrix-assisted refolded on-column with 69% purity, leading to a self-supporting hydrogel at 4 ºC. The gel was washed by using three cycles of PBS and distilled water, which allowed to increase hydrogel purity. This corroborates the fact that hydrogel network is being formed by SD-GFP. The SD peptide was produced in the soluble form in bacterial cells and was purified yielding 90% of purity. The presence of paired cysteines in the SD peptide sequence lead to the formation of disulfide bridges forming tetrameric assemblies. These assemblies present an α-helical structure but are not catalytically active. In the future, the peptide should be in its reduced form (in monomer), so that cysteines can act as a nucleophile, catalyzing the dephosphorylation of pNPP.Pina, AnaRoque, AnaRUNCarvalho, Sara Isabel Santos de2021-10-31T00:30:21Z2018-10-3120182018-10-31T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/52950enginfo: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:RCAAP2024-03-11T04:26:18Zoai:run.unl.pt:10362/52950Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:32:36.706282Repositório 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 |
Nature-based Peptide and Protein Scaffolds as Enzyme Mimetics |
title |
Nature-based Peptide and Protein Scaffolds as Enzyme Mimetics |
spellingShingle |
Nature-based Peptide and Protein Scaffolds as Enzyme Mimetics Carvalho, Sara Isabel Santos de catalysis peptide PTEN scaffold self-assembly Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química |
title_short |
Nature-based Peptide and Protein Scaffolds as Enzyme Mimetics |
title_full |
Nature-based Peptide and Protein Scaffolds as Enzyme Mimetics |
title_fullStr |
Nature-based Peptide and Protein Scaffolds as Enzyme Mimetics |
title_full_unstemmed |
Nature-based Peptide and Protein Scaffolds as Enzyme Mimetics |
title_sort |
Nature-based Peptide and Protein Scaffolds as Enzyme Mimetics |
author |
Carvalho, Sara Isabel Santos de |
author_facet |
Carvalho, Sara Isabel Santos de |
author_role |
author |
dc.contributor.none.fl_str_mv |
Pina, Ana Roque, Ana RUN |
dc.contributor.author.fl_str_mv |
Carvalho, Sara Isabel Santos de |
dc.subject.por.fl_str_mv |
catalysis peptide PTEN scaffold self-assembly Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química |
topic |
catalysis peptide PTEN scaffold self-assembly Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química |
description |
Protein and peptide scaffolds are of interest for many bioengineering applications. The incorporation of catalytic activity in such structures is of major importance, although it represents a challenge. Several strategies are being used to obtain peptide catalysts and protein/peptide-based hydrogels, but the merging of the two fields is still in infancy. This work aims to study a new peptide scaffold, based on the small domain peptide, which is part of PTEN, a tumor suppressor protein. To fully characterize this peptide scaffold, the peptide was produced by two strategies; (i) small domain fused with GFP (SD-GFP) and; (ii) the small domain without fusion partner (SD). Both strategies were developed by using bacterial cells as hosts and purified by chromatographic-based techniques. The soluble SD-GFP scaffold was obtained with 93% purity, and showed catalytic activity towards pNPP, presenting a rate of 5x10-4 s-1, which is an order of magnitude lower than the kcat of PTEN towards pNPP. The production of SD-GFP also led to the formation of inclusion bodies, which were solubilized and matrix-assisted refolded on-column with 69% purity, leading to a self-supporting hydrogel at 4 ºC. The gel was washed by using three cycles of PBS and distilled water, which allowed to increase hydrogel purity. This corroborates the fact that hydrogel network is being formed by SD-GFP. The SD peptide was produced in the soluble form in bacterial cells and was purified yielding 90% of purity. The presence of paired cysteines in the SD peptide sequence lead to the formation of disulfide bridges forming tetrameric assemblies. These assemblies present an α-helical structure but are not catalytically active. In the future, the peptide should be in its reduced form (in monomer), so that cysteines can act as a nucleophile, catalyzing the dephosphorylation of pNPP. |
publishDate |
2018 |
dc.date.none.fl_str_mv |
2018-10-31 2018 2018-10-31T00:00:00Z 2021-10-31T00:30:21Z |
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/10362/52950 |
url |
http://hdl.handle.net/10362/52950 |
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.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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1799137947632533504 |