Expression of the functional carbohydrate-binding module (CBM) of human laforin
Autor(a) principal: | |
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Data de Publicação: | 2010 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/1822/11476 |
Resumo: | Laforin is a human protein associated with the glycogen metabolism, composed of two structurally and functionally independent domains: a phosphatase catalytic domain and a substrate-binding module with glycogen and starch affinity. The main goal of this work is the development of a methodology for the expression of the so far poorly characterized carbohydrate-binding module (CBM) of laforin, allowing its study and development of biomedical applications. The laforin’s CBM sequence was originally cloned by PCR from a human muscle cDNA library. The recombinant protein, containing laforin’s CBM fused to an Arg-Gly-Asp sequence (RGD), was cloned and expressed using vector pET29a and recovered as inclusion bodies (IBs). Refolding of the IBs allowed the purification of soluble, dimeric and functional protein, according to adsorption assays using starch and glycogen. Several other experimental approaches, using both bacteria and yeast, were unsuccessfully tested, pointing towards the difficulties in producing the heterologous protein. Indeed, this is the first work reporting the production of the functional CBM from human laforin. |
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Expression of the functional carbohydrate-binding module (CBM) of human laforinLaforinCarbohydrate-binding moduleRGDInclusion bodiesScience & TechnologyLaforin is a human protein associated with the glycogen metabolism, composed of two structurally and functionally independent domains: a phosphatase catalytic domain and a substrate-binding module with glycogen and starch affinity. The main goal of this work is the development of a methodology for the expression of the so far poorly characterized carbohydrate-binding module (CBM) of laforin, allowing its study and development of biomedical applications. The laforin’s CBM sequence was originally cloned by PCR from a human muscle cDNA library. The recombinant protein, containing laforin’s CBM fused to an Arg-Gly-Asp sequence (RGD), was cloned and expressed using vector pET29a and recovered as inclusion bodies (IBs). Refolding of the IBs allowed the purification of soluble, dimeric and functional protein, according to adsorption assays using starch and glycogen. Several other experimental approaches, using both bacteria and yeast, were unsuccessfully tested, pointing towards the difficulties in producing the heterologous protein. Indeed, this is the first work reporting the production of the functional CBM from human laforin.Fundação para a Ciência e a Tecnologia (FCT, Portugal).ElsevierUniversidade do MinhoMoreira, Susana Margarida GomesCastanheira, PedroCasal, MargaridaFaro, CarlosGama, F. M.20102010-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/1822/11476eng"Protein Expression and Purification". ISSN 1046-5928. 74:2 (2010) 169-174.1046-592810.1016/j.pep.2010.06.01920600946info: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:RCAAP2023-07-21T12:30:13Zoai:repositorium.sdum.uminho.pt:1822/11476Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:25:20.240920Repositório 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 functional carbohydrate-binding module (CBM) of human laforin |
title |
Expression of the functional carbohydrate-binding module (CBM) of human laforin |
spellingShingle |
Expression of the functional carbohydrate-binding module (CBM) of human laforin Moreira, Susana Margarida Gomes Laforin Carbohydrate-binding module RGD Inclusion bodies Science & Technology |
title_short |
Expression of the functional carbohydrate-binding module (CBM) of human laforin |
title_full |
Expression of the functional carbohydrate-binding module (CBM) of human laforin |
title_fullStr |
Expression of the functional carbohydrate-binding module (CBM) of human laforin |
title_full_unstemmed |
Expression of the functional carbohydrate-binding module (CBM) of human laforin |
title_sort |
Expression of the functional carbohydrate-binding module (CBM) of human laforin |
author |
Moreira, Susana Margarida Gomes |
author_facet |
Moreira, Susana Margarida Gomes Castanheira, Pedro Casal, Margarida Faro, Carlos Gama, F. M. |
author_role |
author |
author2 |
Castanheira, Pedro Casal, Margarida Faro, Carlos Gama, F. M. |
author2_role |
author author author author |
dc.contributor.none.fl_str_mv |
Universidade do Minho |
dc.contributor.author.fl_str_mv |
Moreira, Susana Margarida Gomes Castanheira, Pedro Casal, Margarida Faro, Carlos Gama, F. M. |
dc.subject.por.fl_str_mv |
Laforin Carbohydrate-binding module RGD Inclusion bodies Science & Technology |
topic |
Laforin Carbohydrate-binding module RGD Inclusion bodies Science & Technology |
description |
Laforin is a human protein associated with the glycogen metabolism, composed of two structurally and functionally independent domains: a phosphatase catalytic domain and a substrate-binding module with glycogen and starch affinity. The main goal of this work is the development of a methodology for the expression of the so far poorly characterized carbohydrate-binding module (CBM) of laforin, allowing its study and development of biomedical applications. The laforin’s CBM sequence was originally cloned by PCR from a human muscle cDNA library. The recombinant protein, containing laforin’s CBM fused to an Arg-Gly-Asp sequence (RGD), was cloned and expressed using vector pET29a and recovered as inclusion bodies (IBs). Refolding of the IBs allowed the purification of soluble, dimeric and functional protein, according to adsorption assays using starch and glycogen. Several other experimental approaches, using both bacteria and yeast, were unsuccessfully tested, pointing towards the difficulties in producing the heterologous protein. Indeed, this is the first work reporting the production of the functional CBM from human laforin. |
publishDate |
2010 |
dc.date.none.fl_str_mv |
2010 2010-01-01T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/1822/11476 |
url |
http://hdl.handle.net/1822/11476 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
"Protein Expression and Purification". ISSN 1046-5928. 74:2 (2010) 169-174. 1046-5928 10.1016/j.pep.2010.06.019 20600946 |
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 |
Elsevier |
publisher.none.fl_str_mv |
Elsevier |
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 |
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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) |
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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 |
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1799132737520533504 |