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

Authors: Dantas, Giordanni C.; Martins, Paula M. M.; Martins, Daniela A. B.; Gomes, Eleni; Ferreira, Henrique;

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

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.

Country
Brazil
Keywords

Expression vectors, 570, TAP-tag, Citrus, Xanthomonas, Recombinant Fusion Proteins, Genetic Vectors, Genetics and Molecular Microbiology, Microbiology, Open Reading Frames, Bacterial Proteins, Citrus canker, Plant Diseases

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    This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
    3
    popularity
    This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
    Average
    influence
    This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
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    impulse
    This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
    Average
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selected citations
These citations are derived from selected sources.
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
3
Average
Average
Average
Green
gold