Table of Contents
International Journal of Bacteriology
Volume 2013, Article ID 746862, 9 pages
Research Article

Expression, Purification, and Functional Characterization of Atypical Xenocin, Its Immunity Protein, and Their Domains from Xenorhabdus nematophila

School of Biotechnology, Gautam Buddha University, Yamuna Expressway, Greater Noida, Uttar Pradesh 201308, India

Received 30 April 2013; Revised 17 July 2013; Accepted 1 August 2013

Academic Editor: Rodrigo E. Mendes

Copyright © 2013 Jitendra Singh Rathore. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Xenorhabdus nematophila, a gram-negative bacterium belonging to the family Enterobacteriaceae is a natural symbiont of a soil nematode from the family Steinernematidae. In this study cloning, expression, and purification of broad range iron regulated multidomain bacteriocin called xenocin from X. nematophila (66 kDa, encoded by xcinA gene) and its multidomain immunity protein (42 kDa, encoded by ximB gene) have been done. xcinA-ximB (N′ terminal 270 bp), translocation, and translocation-receptor domain of xcinA, ximB, and its hemolysin domain were cloned, expressed, and purified by single step Ni-NTA chromatography under native conditions. In the functional characterization, neutralization of xcinA toxicity by immunity domain of ximB gene was determined by endogenous assay. Exogenous toxic assays results showed that only the purified recombinant xenocin-immunity domain (10 kDa) protein complex had toxic activity. Atypical cognate immunity protein (42 kDa) of xenocin was fusion of immunity domain (10 kDa) and hemolysin domain (32 kDa). In silico analysis of immunity protein revealed its similarity with hemolysin and purine NTPase like proteins. Hemolytic activity was not observed in immunity protein or in its various domains; however, full-length immunity protein lacking Walker motif showed ATPase activity. Finally, using circular dichroism performed secondary structural analyses of all the recombinant proteins/protein complexes.