Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization
Autor(a) principal: | |
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Data de Publicação: | 1989 |
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/10400.1/6143 |
Resumo: | A human liver cDNA library was used to isolate a clone coding for apolipoprotein A-I (Apo A-I). The clone carries the sequence for the prepeptide (18 amino acids), the propeptide (6 amino acids), and the mature protein (243 amino acids). A coding cassette for the proapo A-I molecule was reconstructed by fusing synthetic sequences, chosen to optimize expression and specifying the amino-terminal methionine and amino acids -6 to +14, to a large fragment of the cDNA coding for amino acids 15-243. The module was expressed in pOTS-Nco, an Escherichia coli expression vector carrying the regulatable X P^ promoter, leading to the production of proapolipoprotein A-I at up to 10% of total soluble proteins. The recombinant polypeptide was purified and characterized in terms of apparent molecular mass, isoelectric point, and by both chemical and enzymatic peptide mapping. In addition, it was assayed in vitro for the stimulation of the enzyme lecithin: cholesterol acyltransferase. The data show for the first time that proapo A-I can be produced efficiently in E. coli as a stable and undegraded protein having physical and functional properties indistinguishable from those of the natural product. |
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Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterizationA human liver cDNA library was used to isolate a clone coding for apolipoprotein A-I (Apo A-I). The clone carries the sequence for the prepeptide (18 amino acids), the propeptide (6 amino acids), and the mature protein (243 amino acids). A coding cassette for the proapo A-I molecule was reconstructed by fusing synthetic sequences, chosen to optimize expression and specifying the amino-terminal methionine and amino acids -6 to +14, to a large fragment of the cDNA coding for amino acids 15-243. The module was expressed in pOTS-Nco, an Escherichia coli expression vector carrying the regulatable X P^ promoter, leading to the production of proapolipoprotein A-I at up to 10% of total soluble proteins. The recombinant polypeptide was purified and characterized in terms of apparent molecular mass, isoelectric point, and by both chemical and enzymatic peptide mapping. In addition, it was assayed in vitro for the stimulation of the enzyme lecithin: cholesterol acyltransferase. The data show for the first time that proapo A-I can be produced efficiently in E. coli as a stable and undegraded protein having physical and functional properties indistinguishable from those of the natural product.Mary Ann Liebert, Inc., PublishersSapientiaMoguilevsky, N.Roobol, C.Loriau, R.Guillaume, J. P.Jacobs, P.Cravador, A.Herzog, A.Brouwers, L.Scarso, A.Gilles, P.Homquist, L.Carlson, L. A.Bollen, A.2015-06-15T09:15:53Z19891989-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10400.1/6143eng0198-0238AUT: ACR00659;info: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:RCAAP2023-07-24T10:17:24Zoai:sapientia.ualg.pt:10400.1/6143Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:59:00.919406Repositório 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 |
Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization |
title |
Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization |
spellingShingle |
Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization Moguilevsky, N. |
title_short |
Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization |
title_full |
Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization |
title_fullStr |
Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization |
title_full_unstemmed |
Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization |
title_sort |
Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization |
author |
Moguilevsky, N. |
author_facet |
Moguilevsky, N. Roobol, C. Loriau, R. Guillaume, J. P. Jacobs, P. Cravador, A. Herzog, A. Brouwers, L. Scarso, A. Gilles, P. Homquist, L. Carlson, L. A. Bollen, A. |
author_role |
author |
author2 |
Roobol, C. Loriau, R. Guillaume, J. P. Jacobs, P. Cravador, A. Herzog, A. Brouwers, L. Scarso, A. Gilles, P. Homquist, L. Carlson, L. A. Bollen, A. |
author2_role |
author author author author author author author author author author author author |
dc.contributor.none.fl_str_mv |
Sapientia |
dc.contributor.author.fl_str_mv |
Moguilevsky, N. Roobol, C. Loriau, R. Guillaume, J. P. Jacobs, P. Cravador, A. Herzog, A. Brouwers, L. Scarso, A. Gilles, P. Homquist, L. Carlson, L. A. Bollen, A. |
description |
A human liver cDNA library was used to isolate a clone coding for apolipoprotein A-I (Apo A-I). The clone carries the sequence for the prepeptide (18 amino acids), the propeptide (6 amino acids), and the mature protein (243 amino acids). A coding cassette for the proapo A-I molecule was reconstructed by fusing synthetic sequences, chosen to optimize expression and specifying the amino-terminal methionine and amino acids -6 to +14, to a large fragment of the cDNA coding for amino acids 15-243. The module was expressed in pOTS-Nco, an Escherichia coli expression vector carrying the regulatable X P^ promoter, leading to the production of proapolipoprotein A-I at up to 10% of total soluble proteins. The recombinant polypeptide was purified and characterized in terms of apparent molecular mass, isoelectric point, and by both chemical and enzymatic peptide mapping. In addition, it was assayed in vitro for the stimulation of the enzyme lecithin: cholesterol acyltransferase. The data show for the first time that proapo A-I can be produced efficiently in E. coli as a stable and undegraded protein having physical and functional properties indistinguishable from those of the natural product. |
publishDate |
1989 |
dc.date.none.fl_str_mv |
1989 1989-01-01T00:00:00Z 2015-06-15T09:15:53Z |
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/10400.1/6143 |
url |
http://hdl.handle.net/10400.1/6143 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
0198-0238 AUT: ACR00659; |
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 |
Mary Ann Liebert, Inc., Publishers |
publisher.none.fl_str_mv |
Mary Ann Liebert, Inc., Publishers |
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) |
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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1799133210578255872 |