High expression of human carboxypeptidase M in Pichia pastoris. Purification and partial characterization
Autor(a) principal: | |
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Data de Publicação: | 2006 |
Outros Autores: | , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNIFESP |
dARK ID: | ark:/48912/001300001b333 |
Texto Completo: | http://dx.doi.org/10.1590/S0100-879X2006000200007 http://repositorio.unifesp.br/handle/11600/28713 |
Resumo: | Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidylinositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. the present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. the cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. the cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. the recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. the enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein(-1) min(-1)). This purification protocol permitted us to obtain 410 mg of the purified protein per titer of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes. |
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High expression of human carboxypeptidase M in Pichia pastoris. Purification and partial characterizationkininshuman carboxypeptidase Mrecombinant proteinPichia pastorisCarboxypeptidase M (CPM) is an extracellular glycosylphosphatidylinositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. the present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. the cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. the cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. the recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. the enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein(-1) min(-1)). This purification protocol permitted us to obtain 410 mg of the purified protein per titer of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes.Universidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Nefrol, BR-04023062 São Paulo, BrazilUniv Mogi Cruzes, São Paulo, BrazilUniv Fed Minas Gerais, Dept Fisiol & Biofis, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Nefrol, BR-04023062 São Paulo, BrazilWeb of ScienceAssoc Bras Divulg CientificaUniversidade Federal de São Paulo (UNIFESP)Univ Mogi CruzesUniversidade Federal de Minas Gerais (UFMG)Craveiro, Rogerio Bastos [UNIFESP]Ramalho, João Daivison Silva [UNIFESP]Chagas, Jair Ribeiro [UNIFESP]Wang, Pamella Huey Mei [UNIFESP]Casarini, Dulce Elena [UNIFESP]Pesquero, Jorge Luiz [UNIFESP]Araujo, Ronaldo Carvalho [UNIFESP]Pesquero, João Bosco [UNIFESP]2016-01-24T12:40:56Z2016-01-24T12:40:56Z2006-02-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersion211-217application/pdfhttp://dx.doi.org/10.1590/S0100-879X2006000200007Brazilian Journal of Medical and Biological Research. São Paulo: Assoc Bras Divulg Cientifica, v. 39, n. 2, p. 211-217, 2006.10.1590/S0100-879X20060002000070100-879XS0100-879X2006000200007http://repositorio.unifesp.br/handle/11600/28713WOS:000235452400007ark:/48912/001300001b333engBrazilian Journal of Medical and Biological Researchinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESP2024-07-30T22:43:55Zoai:repositorio.unifesp.br/:11600/28713Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestbiblioteca.csp@unifesp.bropendoar:34652024-12-11T21:06:54.476740Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false |
dc.title.none.fl_str_mv |
High expression of human carboxypeptidase M in Pichia pastoris. Purification and partial characterization |
title |
High expression of human carboxypeptidase M in Pichia pastoris. Purification and partial characterization |
spellingShingle |
High expression of human carboxypeptidase M in Pichia pastoris. Purification and partial characterization Craveiro, Rogerio Bastos [UNIFESP] kinins human carboxypeptidase M recombinant protein Pichia pastoris |
title_short |
High expression of human carboxypeptidase M in Pichia pastoris. Purification and partial characterization |
title_full |
High expression of human carboxypeptidase M in Pichia pastoris. Purification and partial characterization |
title_fullStr |
High expression of human carboxypeptidase M in Pichia pastoris. Purification and partial characterization |
title_full_unstemmed |
High expression of human carboxypeptidase M in Pichia pastoris. Purification and partial characterization |
title_sort |
High expression of human carboxypeptidase M in Pichia pastoris. Purification and partial characterization |
author |
Craveiro, Rogerio Bastos [UNIFESP] |
author_facet |
Craveiro, Rogerio Bastos [UNIFESP] Ramalho, João Daivison Silva [UNIFESP] Chagas, Jair Ribeiro [UNIFESP] Wang, Pamella Huey Mei [UNIFESP] Casarini, Dulce Elena [UNIFESP] Pesquero, Jorge Luiz [UNIFESP] Araujo, Ronaldo Carvalho [UNIFESP] Pesquero, João Bosco [UNIFESP] |
author_role |
author |
author2 |
Ramalho, João Daivison Silva [UNIFESP] Chagas, Jair Ribeiro [UNIFESP] Wang, Pamella Huey Mei [UNIFESP] Casarini, Dulce Elena [UNIFESP] Pesquero, Jorge Luiz [UNIFESP] Araujo, Ronaldo Carvalho [UNIFESP] Pesquero, João Bosco [UNIFESP] |
author2_role |
author author author author author author author |
dc.contributor.none.fl_str_mv |
Universidade Federal de São Paulo (UNIFESP) Univ Mogi Cruzes Universidade Federal de Minas Gerais (UFMG) |
dc.contributor.author.fl_str_mv |
Craveiro, Rogerio Bastos [UNIFESP] Ramalho, João Daivison Silva [UNIFESP] Chagas, Jair Ribeiro [UNIFESP] Wang, Pamella Huey Mei [UNIFESP] Casarini, Dulce Elena [UNIFESP] Pesquero, Jorge Luiz [UNIFESP] Araujo, Ronaldo Carvalho [UNIFESP] Pesquero, João Bosco [UNIFESP] |
dc.subject.por.fl_str_mv |
kinins human carboxypeptidase M recombinant protein Pichia pastoris |
topic |
kinins human carboxypeptidase M recombinant protein Pichia pastoris |
description |
Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidylinositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. the present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. the cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. the cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. the recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. the enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein(-1) min(-1)). This purification protocol permitted us to obtain 410 mg of the purified protein per titer of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes. |
publishDate |
2006 |
dc.date.none.fl_str_mv |
2006-02-01 2016-01-24T12:40:56Z 2016-01-24T12:40:56Z |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1590/S0100-879X2006000200007 Brazilian Journal of Medical and Biological Research. São Paulo: Assoc Bras Divulg Cientifica, v. 39, n. 2, p. 211-217, 2006. 10.1590/S0100-879X2006000200007 0100-879X S0100-879X2006000200007 http://repositorio.unifesp.br/handle/11600/28713 WOS:000235452400007 |
dc.identifier.dark.fl_str_mv |
ark:/48912/001300001b333 |
url |
http://dx.doi.org/10.1590/S0100-879X2006000200007 http://repositorio.unifesp.br/handle/11600/28713 |
identifier_str_mv |
Brazilian Journal of Medical and Biological Research. São Paulo: Assoc Bras Divulg Cientifica, v. 39, n. 2, p. 211-217, 2006. 10.1590/S0100-879X2006000200007 0100-879X S0100-879X2006000200007 WOS:000235452400007 ark:/48912/001300001b333 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Brazilian Journal of Medical and Biological Research |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
211-217 application/pdf |
dc.publisher.none.fl_str_mv |
Assoc Bras Divulg Cientifica |
publisher.none.fl_str_mv |
Assoc Bras Divulg Cientifica |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UNIFESP instname:Universidade Federal de São Paulo (UNIFESP) instacron:UNIFESP |
instname_str |
Universidade Federal de São Paulo (UNIFESP) |
instacron_str |
UNIFESP |
institution |
UNIFESP |
reponame_str |
Repositório Institucional da UNIFESP |
collection |
Repositório Institucional da UNIFESP |
repository.name.fl_str_mv |
Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP) |
repository.mail.fl_str_mv |
biblioteca.csp@unifesp.br |
_version_ |
1818602604712689664 |