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

Texto completo
Autor(es):
Soares-Costa, A. [1] ; Silveira, R. S. [1] ; Novo, M. T. M. [2] ; Alves, M. F. M. [3] ; Carmona, A. K. [3] ; Belasque, Jr., J. [4] ; Henrique-Silva, F. [1]
Número total de Autores: 7
Afiliação do(s) autor(es):
[1] Univ Fed Sao Carlos, Mol Biol Lab, Dept Genet & Evolucao, BR-13560 Sao Carlos, SP - Brazil
[2] Univ Fed Sao Carlos, Lab Bioquim & Biol Mol Microrganismos, Dept Genet & Evolucao, BR-13560 Sao Carlos, SP - Brazil
[3] Univ Fed Sao Paulo, Dept Biofis, Sao Paulo - Brazil
[4] Fundo Def Citricultura, Araraquara, SP - Brazil
Número total de Afiliações: 4
Tipo de documento: Artigo Científico
Fonte: Genetics and Molecular Research; v. 11, n. 4, p. 4043-4057, 2012.
Citações Web of Science: 3
Resumo

Xanthomonas 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. (AU)

Processo FAPESP: 98/14138-2 - Center for Structural Molecular Biotechnology
Beneficiário:Glaucius Oliva
Linha de fomento: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
Processo FAPESP: 05/59833-5 - Desenvolvimento de canas-de-açúcar transgênicas superexpressando cistatinas visando o aumento da resistência a insetos
Beneficiário:Andrea Soares da Costa Fuentes
Linha de fomento: Bolsas no Brasil - Pós-Doutorado