High expression of human carboxypeptidase M in Pichia pastoris. Purification and partial characterization

Detalhes bibliográficos
Autor(a) principal: Craveiro, Rogerio Bastos [UNIFESP]
Data de Publicação: 2006
Outros Autores: 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]
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.
id UFSP_dc1059d7520ef39ee5a6c10fa5f5169a
oai_identifier_str oai:repositorio.unifesp.br/:11600/28713
network_acronym_str UFSP
network_name_str Repositório Institucional da UNIFESP
repository_id_str 3465
spelling 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