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

作者：Wang, Yu; Gong, Guo-Hua; Zhou, Wei; Zhang, Bin; Bao, Shu-Yin; Wei, Cheng-Xi; Yue, Jun-Jie^*; Zhang, Yan-Fen

来源：Molecules, 2014, 19(11): 18090-18101.

DOI：10.3390/molecules191118090

• VirG
• apyrase
• pull-down

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 M90T Delta phoN2/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 alpha-domain of VirG to influence the function of VirG.

• 出版日期2014-11
• 单位内蒙古民族大学; 通辽市医院; 中国人民解放军军事医学科学院