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Molecules 2014, 19(11), 18090-18101;

Analysis on the Interaction Domain of VirG and Apyrase by Pull-Down Assay

Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for Nationalities, Tongliao 028000, China
State Key Laboratory of Medical Microbiology and Biosafety, Academy of Military Medical Sciences, Biotechnology Institute of Beijing, Beijing 100071, China
Affiliated Hospital of Inner Mongolia University for Nationalities, Tongliao 028000, China
Tongliao Hospital, Tongliao 028000, China
These authors contributed equally to this work.
Authors to whom correspondence should be addressed.
Received: 10 September 2014 / Revised: 19 October 2014 / Accepted: 21 October 2014 / Published: 5 November 2014
(This article belongs to the Section Molecular Diversity)
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VirG is outer membrane protein of Shigella and affects the spread of Shigella. Recently it has been reported that apyrase influences the location of VirG, although the underlying mechanism remains poorly understood. The site of interaction between apyrase and VirG is the focus of our research. First we constructed recombinant plasmid pHIS-phoN2 and pS-(v1–1102, v53–758, v759–1102, v53–319, v320–507, v507–758) by denaturation-renaturation, the phoN2:kan mutant of Shigella flexneri 5a M90T by a modified version of the lambda red recombination protocol originally described by Datsenko and Wanner and the complemented strain M90TΔphoN2/pET24a(PhisphoN2). Second, the recombinant plasmid pHIS-phoN2 and the pS-(v1–1102, v53–758, v759–1102, v53–319, v320–507, v507–758) were transformed into E. coli BL21 (DE3) and induced to express the fusion proteins. Third, the fusion proteins were purified and the interaction of VirG and apyrase was identified by pull-down. Fourth, VirG was divided and the interaction site of apyrase and VirG was determined. Finally, how apyrase affects the function of VirG was analyzed by immunofluorescence. Accordingly, the results provided the data supporting the fact that apyrase combines with the α-domain of VirG to influence the function of VirG. View Full-Text
Keywords: VirG; apyrase; pull-down VirG; apyrase; pull-down

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Wang, Y.; Gong, G.-H.; Zhou, W.; Zhang, B.; Bao, S.-Y.; Wei, C.-X.; Yue, J.-J.; Zhang, Y.-F. Analysis on the Interaction Domain of VirG and Apyrase by Pull-Down Assay. Molecules 2014, 19, 18090-18101.

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