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.

Anti-Metalloprotease P-I Single-Domain Antibodies: Tools for Next-Generation Snakebite Antivenoms

“…Subsequently, the culture was centrifuged for 15 min at 4,500 rpm at room temperature and the bacterial pellet was resuspended in 25 mL of lysis buffer (10 mM Tris base, 1 M NaCl, Triton X-100 (0.05%, pH 8.0)). Cellular contents were lysed on ice through 2 cycles of ultrasonication (Vibracell ® VC100, Sonic and Materials Inc.) with an interval of 30 s, adapted from Dantas et al [ 37 ]. Bacterial lysates were centrifuged for 15 min at 10,500 rpm in order to separate soluble contents from insoluble cell debris.…”