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

Detalhes bibliográficos
Autor(a) principal: Craveiro,R.B.
Data de Publicação: 2006
Outros Autores: Ramalho,J.D., Chagas,J.R., Wang,P.H.M., Casarini,D.E., Pesquero,J.L., Araújo,R.C., Pesquero,J.B.
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Brazilian Journal of Medical and Biological Research
Texto Completo: http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2006000200007
Resumo: Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-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 liter 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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spelling High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterizationKininsHuman carboxypeptidase MRecombinant proteinPichia pastorisCarboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-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 liter 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 processesAssociação Brasileira de Divulgação Científica2006-02-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2006000200007Brazilian Journal of Medical and Biological Research v.39 n.2 2006reponame:Brazilian Journal of Medical and Biological Researchinstname:Associação Brasileira de Divulgação Científica (ABDC)instacron:ABDC10.1590/S0100-879X2006000200007info:eu-repo/semantics/openAccessCraveiro,R.B.Ramalho,J.D.Chagas,J.R.Wang,P.H.M.Casarini,D.E.Pesquero,J.L.Araújo,R.C.Pesquero,J.B.eng2006-05-04T00:00:00Zoai:scielo:S0100-879X2006000200007Revistahttps://www.bjournal.org/https://old.scielo.br/oai/scielo-oai.phpbjournal@terra.com.br||bjournal@terra.com.br1414-431X0100-879Xopendoar:2006-05-04T00:00Brazilian Journal of Medical and Biological Research - Associação Brasileira de Divulgação Científica (ABDC)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,R.B.
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,R.B.
author_facet Craveiro,R.B.
Ramalho,J.D.
Chagas,J.R.
Wang,P.H.M.
Casarini,D.E.
Pesquero,J.L.
Araújo,R.C.
Pesquero,J.B.
author_role author
author2 Ramalho,J.D.
Chagas,J.R.
Wang,P.H.M.
Casarini,D.E.
Pesquero,J.L.
Araújo,R.C.
Pesquero,J.B.
author2_role author
author
author
author
author
author
author
dc.contributor.author.fl_str_mv Craveiro,R.B.
Ramalho,J.D.
Chagas,J.R.
Wang,P.H.M.
Casarini,D.E.
Pesquero,J.L.
Araújo,R.C.
Pesquero,J.B.
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 glycosylphosphatidyl-inositol-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 liter 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
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://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2006000200007
url http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2006000200007
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 10.1590/S0100-879X2006000200007
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv text/html
dc.publisher.none.fl_str_mv Associação Brasileira de Divulgação Científica
publisher.none.fl_str_mv Associação Brasileira de Divulgação Científica
dc.source.none.fl_str_mv Brazilian Journal of Medical and Biological Research v.39 n.2 2006
reponame:Brazilian Journal of Medical and Biological Research
instname:Associação Brasileira de Divulgação Científica (ABDC)
instacron:ABDC
instname_str Associação Brasileira de Divulgação Científica (ABDC)
instacron_str ABDC
institution ABDC
reponame_str Brazilian Journal of Medical and Biological Research
collection Brazilian Journal of Medical and Biological Research
repository.name.fl_str_mv Brazilian Journal of Medical and Biological Research - Associação Brasileira de Divulgação Científica (ABDC)
repository.mail.fl_str_mv bjournal@terra.com.br||bjournal@terra.com.br
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