Recombinant expression and characterization of a cysteine peptidase from Xanthomonas citri subsp citri

Página do item simplificado
dc.contributor.authorSoares-Costa, Andrea
dc.contributor.authorSilveira, Roseli Santos da
dc.contributor.authorNovo, Maria Teresa Marques
dc.contributor.authorAlves, Marcio Fernando Madureira [UNIFESP]
dc.contributor.authorCarmona, Adriana Karaoglanovic [UNIFESP]
dc.contributor.authorBelasque Junior, José
dc.contributor.authorHenrique-Silva, Flávio
dc.contributor.institutionUniversidade Federal de São Carlos (UFSCar)
dc.contributor.institutionUniversidade Federal de São Paulo (UNIFESP)
dc.contributor.institutionFundo Def Citricultura
dc.date.accessioned2016-01-24T14:17:39Z
dc.date.available2016-01-24T14:17:39Z
dc.date.issued2012-01-01
dc.description.abstractXanthomonas citri subsp citri (Xac) is the bacterium responsible for citrus canker disease in citrus plants. the aim of this study was to describe the recombinant expression, purification, and characterization of a cysteine peptidase from Xac strain 306, which is a candidate for involvement in the pathogenicity of this bacterium. the gene was cloned and expressed in Pichia pastoris, and the cysteine peptidase was successfully expressed, secreted, and purified using affinity chromatography with a yield of approximately 10 mg/L. A polyclonal antibody produced against cysteine peptidase from X. citri subsp citri fused with HIS tag ((HIS)CPXAC) recognized the purified recombinant cysteine peptidase (HIS)CPXAC, confirming the correct production of this protein in P. pastoris. the same antibody detected the protein in the culture supernatant of Xac grown in pathogenicity-inducing medium. Kinetic analysis revealed that (HIS)CPXAC hydrolyzed the carbobenzoxy-Leu-Arg-7-amido-4-methylcoumarin substrate with a catalytic efficiency (k(cat)/K-m) of 47 mu M-1.s(-1). the purified (HIS)CPXAC displayed maximal catalytic activity at pH 5.5 and 30 degrees C. the recombinant enzyme was inhibited by the specific cysteine peptidase inhibitor E-64, as well as by the recombinant cysteine peptidase inhibitors CaneCPI-1, CaneCPI-2, CaneCPI-3, and CaneCPI-4, with K-i values of 1.214, 84.64, 0.09, 0.09, and 0.012 nM, respectively. Finally, the N-terminal sequencing of the purified protein enabled the identification of the first 5 amino acid residues (AVHGM) immediately after the putative signal peptide, thereby enabling the identification of the cleavage point and corroborating previous studies that have identified this sequence in a secreted protein from Xanthomonas spp.en
dc.description.affiliationUniv Fed Sao Carlos, Mol Biol Lab, Dept Genet & Evolucao, BR-13560 Sao Carlos, SP, Brazil
dc.description.affiliationUniv Fed Sao Carlos, Lab Bioquim & Biol Mol Microrganismos, Dept Genet & Evolucao, BR-13560 Sao Carlos, SP, Brazil
dc.description.affiliationUniversidade Federal de São Paulo, Dept Biofis, São Paulo, Brazil
dc.description.affiliationFundo Def Citricultura, Araraquara, SP, Brazil
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Dept Biofis, São Paulo, Brazil
dc.description.sourceWeb of Science
dc.description.sponsorshipFundo de Defesa da Citricultura
dc.description.sponsorshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.description.sponsorshipIDFAPESP: 98/14138-2
dc.description.sponsorshipIDFAPESP: 05/59833-5
dc.format.extent4043-4057
dc.identifierhttp://dx.doi.org/10.4238/2012.November.28.3
dc.identifier.citationGenetics and Molecular Research. Ribeirao Preto: Funpec-editora, v. 11, n. 4, p. 4043-4057, 2012.
dc.identifier.doi10.4238/2012.November.28.3
dc.identifier.issn1676-5680
dc.identifier.urihttp://repositorio.unifesp.br/handle/11600/34407
dc.identifier.wosWOS:000313960500050
dc.language.isoeng
dc.publisherFunpec-editora
dc.relation.ispartofGenetics and Molecular Research
dc.rightsinfo:eu-repo/semantics/openAccess
dc.subjectCysteine peptidaseen
dc.subjectXanthomonas citri subsp citrien
dc.subjectPichia pastorisen
dc.subjectRecombinant expressionen
dc.titleRecombinant expression and characterization of a cysteine peptidase from Xanthomonas citri subsp citrien
dc.typeinfo:eu-repo/semantics/article
Arquivos
Coleções
Em verificação - Geral