A protein expression system for tandem affinity purification in Xanthomonas citri subsp. citri

Autor(es)

Giordanni C. Dantas ^[1] ; Paula M.M. Martins ^[2] ; Daniela A.B. Martins ^[3] ; Eleni Gomes ^[4] ; Henrique Ferreira ^[5]

Afiliação do(s) autor(es)

^[1] Universidade Estadual Paulista. Instituto de Biociências. Depto. de Bioquimica e Microbiologia - Brasil

^[2] Universidade Estadual Paulista. Instituto de Biociências. Depto. de Bioquimica e Microbiologia - Brasil

^[3] Universidade Estadual Paulista. Instituto de Química. Depto. de Bioquímica e Tecnologia Química - Brasil

^[4] Universidade Estadual Paulista. Depto. de Biologia - Brasil

^[5] Universidade Estadual Paulista. Instituto de Biociências. Depto. de Bioquimica e Microbiologia - Brasil

• Tipo de documento: Artigo Científico
• Fonte: Brazilian Journal of Microbiology; v. 47, n. 2, p. 518-526, 2016-06-00.

Resumo

Abstract Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo. (AU)

• Processo FAPESP: 13/19806-5 - Seleção de sítio de divisão em Xanthomonas citri subsp. citri: caracterização de minCD
• Beneficiário: Giordanni Cabral Dantas
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 04/09173-6 - Caracterização funcional de proteínas hipotéticas de bactérias fitopatogênicas
• Beneficiário: Henrique Ferreira
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores
• Processo FAPESP: 06/59494-9 - Localizacao sub-celular de proteinas marcadas com gfp em xanthomonas axonopodis pv. citri por microscopia de fluorescencia.
• Beneficiário: Paula Maria Moreira Martins
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 13/50367-8 - New environmental-friendly compounds to combat Citrus canker
• Beneficiário: Henrique Ferreira
• Modalidade de apoio: Auxílio à Pesquisa - Regular