Clonagem e expressão do gene da tiorredoxina 1 de Paracoccidioides brasiliensis em Pichia pastoris / Cloning and expression of the thioredoxin 1 gene of Paracoccidioides brasiliensis in pichia pastoris

AUTOR(ES)

Lorena Cardoso Cintra

FONTE

IBICT - Instituto Brasileiro de Informação em Ciência e Tecnologia

DATA DE PUBLICAÇÃO

27/08/2010

RESUMO

The termodimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a human systemic mycosis of high prevalence in Latin America. P. brasiliensis is exposed to oxidative stress (OS) caused by reactive oxygen species (ROS) produced by the defense cells of the human host. When the invasion by pathogens occurs, the host defense system generates ROS to fight the invader. Inside the human host, P. brasiliensis is phagocytosed by macrophages, facing an extremely hostile environment due to nitric oxide and hydrogen peroxide. The Trx1 is an intracellular redox protein, which participates in the maintenance of cell redox homeostasis, both in terms of OS as reducer. It is ubiquitous and is characterized by typical CXXC active site, responsible for oxidation, reduction, or isomerization of proteins disulfide bonds. In a previous work, it was isolated, characterized and cloned into expression vector pGEX-4T-3 cDNA coding for TRX1 of P. brasiliensis (accession number AY376435). The recombinant protein (recPbTRX1) was produced and partially purified and the yeast cells of P. brasiliensis showed increased expression of the gene coding for PbTRX1 in response to OS. This study aimed the heterologous expression of cDNA of a thioredoxin of the fungus P. brasiliensis in Pichia pastoris, in order to obtain it in larger amounts for their subsequent biochemical characterization and application in biotechnological processes. The P. brasiliensis thioredoxin 1 (trx1) cDNA was obtained via PCR using the plasmid pGEX-Trx1 as template and cloned into expression vector pHIL-D2 and pPIC9 (for intracellular and extracellular expression). The insertion of the interested gene in the correct orientation was verified by sequencing and the homology was observed with Trx1 P. brasiliensis. These vectors were used to transform the P. pastoris yeast strain SMD1168 with his4- genotype. The presence of the cassettes expression was confirmed in the yeasts genome. No transformants able to secrete the protein from the building with the vector pPIC9 were detected and the intracellular production was carried from the pHIL-D2 vector.

ASSUNTO(S)

clonagem expressão heteróloga tiorredoxina 1 pichia pastoris ciencias biologicas paracoccidioides brasiliensis - pichia pastoris - análise; paracoccidioidomicose; levedos cloning heterologous expression thioredoxin 1 pichia pastoris

Documentos Relacionados

• Caracterização Molecular e Expressão Heteróloga de um cDNA Codificante para Tiorredoxina do fungo patogênico humano Paracoccidioides brasiliensis
• Caracterização Molecular e Expressão Heteróloga de um cDNA Codificante para Tiorredoxina do fungo patogênico humano Paracoccidioides brasiliensis
• CLONAGEM, CARACTERIZAÇÃO E EXPRESSÃO HETERÓLOGA DE UMA CALNEXINA HOMÓLOGA DO FUNGO PATOGÊNICO Paracoccidioides brasiliensis
• Clonagem e expressão de fragmentos de anticorpos (FABs) anti-HIV-1 em Pichia pastoris
• Clonagem e expressão em Pichia pastoris da forma truncada da glicoproteína D (gD) de Herpesvírus Bovino tipo 5