title: Recombinant expression and characterization of a  cysteine peptidase from Xanthomonas citri subsp citri.
abstract: 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 μM(-1)∙s(-1). The purified ((HIS))CPXAC displayed maximal catalytic activity at pH 5.5 and 30°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.
authors.affiliation: Laboratório de Biologia Molecular, Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, Brasil.
authors.forename: A
authors.initials: A
authors.lastname: Soares-Costa
authors.forename: R S
authors.initials: RS
authors.lastname: Silveira
authors.forename: M T M
authors.initials: MT
authors.lastname: Novo
authors.forename: M F M
authors.initials: MF
authors.lastname: Alves
authors.forename: A K
authors.initials: AK
authors.lastname: Carmona
authors.forename: J
authors.initials: J
authors.lastname: Belasque
authors.forename: F
authors.initials: F
authors.lastname: Henrique-Silva
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis / Q000187:drug effects; D019295:Computational Biology; D003470:Culture Media; D057056:Cysteine Proteases / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013696:Temperature; D014974:Xanthomonas / Q000187:drug effects / Q000201:enzymology* / Q000472:pathogenicity
chemical_list: D003470:Culture Media; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D057056:Cysteine Proteases