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

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dc.contributor.authorCraveiro, Rogerio Bastos [UNIFESP]
dc.contributor.authorRamalho, João Daivison Silva [UNIFESP]
dc.contributor.authorChagas, Jair Ribeiro [UNIFESP]
dc.contributor.authorWang, Pamella Huey Mei [UNIFESP]
dc.contributor.authorCasarini, Dulce Elena [UNIFESP]
dc.contributor.authorPesquero, Jorge Luiz [UNIFESP]
dc.contributor.authorAraujo, Ronaldo Carvalho [UNIFESP]
dc.contributor.authorPesquero, João Bosco [UNIFESP]
dc.contributor.institutionUniversidade Federal de São Paulo (UNIFESP)
dc.contributor.institutionUniv Mogi Cruzes
dc.contributor.institutionUniversidade Federal de Minas Gerais (UFMG)
dc.date.accessioned2016-01-24T12:40:56Z
dc.date.available2016-01-24T12:40:56Z
dc.date.issued2006-02-01
dc.description.abstractCarboxypeptidase 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.en
dc.description.affiliationUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04023062 São Paulo, Brazil
dc.description.affiliationUniversidade Federal de São Paulo, Escola Paulista Med, Dept Nefrol, BR-04023062 São Paulo, Brazil
dc.description.affiliationUniv Mogi Cruzes, São Paulo, Brazil
dc.description.affiliationUniv Fed Minas Gerais, Dept Fisiol & Biofis, Belo Horizonte, MG, Brazil
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04023062 São Paulo, Brazil
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Escola Paulista Med, Dept Nefrol, BR-04023062 São Paulo, Brazil
dc.description.sourceWeb of Science
dc.format.extent211-217
dc.identifierhttp://dx.doi.org/10.1590/S0100-879X2006000200007
dc.identifier.citationBrazilian Journal of Medical and Biological Research. São Paulo: Assoc Bras Divulg Cientifica, v. 39, n. 2, p. 211-217, 2006.
dc.identifier.doi10.1590/S0100-879X2006000200007
dc.identifier.issn0100-879X
dc.identifier.scieloS0100-879X2006000200007
dc.identifier.urihttp://repositorio.unifesp.br/handle/11600/28713
dc.identifier.wosWOS:000235452400007
dc.language.isoeng
dc.publisherAssoc Bras Divulg Cientifica
dc.relation.ispartofBrazilian Journal of Medical and Biological Research
dc.rightsinfo:eu-repo/semantics/openAccess
dc.subjectkininsen
dc.subjecthuman carboxypeptidase Men
dc.subjectrecombinant proteinen
dc.subjectPichia pastorisen
dc.titleHigh expression of human carboxypeptidase M in Pichia pastoris. Purification and partial characterizationen
dc.typeinfo:eu-repo/semantics/article
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