Navegando por Palavras-chave "Recombinant expression"

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    Biochemical characterization of a protein tyrosine phosphatase from Trypanosoma cruzi involved in metacyclogenesis and cell invasion
    (Elsevier B.V., 2011-05-13) Gallo, Gloria [UNIFESP]; Ramos, Thiago Cesar Prata [UNIFESP]; Tavares, Fernanda; Rocha, Antonio Augusto [UNIFESP]; Machi, Emerson; Schenkman, Sergio [UNIFESP]; Bahia, Diana [UNIFESP]; Pesquero, João Bosco [UNIFESP]; Wuertele, Martin [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Ctr Biol Mol Estrutural
    Protein tyrosine phosphatases (PTPs) form a large family of enzymes involved in the regulation of numerous cellular functions in eukaryotes. Several protein tyrosine phosphatases have been recently identified in trypanosomatides. Here we report the purification and biochemical characterization of TcPTP1, a protein tyrosine phosphatase from Trypanosoma cruzi, the causing agent of Chagas' disease. the enzyme was cloned and expressed recombinantly in Escherichia coli and purified by Ni-affinity chromatography. Biochemical characterization of recombinant TcPTP1 with the PTP pseudo-substrate pNPP allowed the estimation of a Michaelis-Menten constant K-m of 4.5 mM and a k(cat) of 2.8 s(-1). We were able to demonstrate inhibition of the enzyme by the PTP1b inhibitor BZ3, which on its turn was able to accelerate the differentiation of epimastigotes into metacyclic forms of T. cruzi induced by nutritional stress. Additionally, this compound was able to inhibit by 50% the infectivity of T. cruzi trypomastigotes in a separate cellular assay. in conclusion our results indicate that TcPTP1 is of importance for cellular differentiation and invasivity of this parasite and thus is a valid target for the rational drug design of potential antibiotics directed against T. cruzi. (C) 2011 Elsevier Inc. All rights reserved.
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    Recombinant expression and characterization of a cysteine peptidase from Xanthomonas citri subsp citri
    (Funpec-editora, 2012-01-01) Soares-Costa, Andrea; Silveira, Roseli Santos da; Novo, Maria Teresa Marques; Alves, Marcio Fernando Madureira [UNIFESP]; Carmona, Adriana Karaoglanovic [UNIFESP]; Belasque Junior, José; Henrique-Silva, Flávio; Universidade Federal de São Carlos (UFSCar); Universidade Federal de São Paulo (UNIFESP); Fundo Def Citricultura
    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.