Analysis on the interaction domain of VirG and apyrase by pull-down assay.
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.
Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for Nationalities, Tongliao 028000, China.
Yu
Y
Wang
Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for Nationalities, Tongliao 028000, China.
Guo-Hua
GH
Gong
State Key Laboratory of Medical Microbiology and Biosafety, Academy of Military Medical Sciences, Biotechnology Institute of Beijing, Beijing 100071, China.
Wei
W
Zhou
Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for Nationalities, Tongliao 028000, China.
Bin
B
Zhang
Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for Nationalities, Tongliao 028000, China.
Shu-Yin
SY
Bao
Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for Nationalities, Tongliao 028000, China.
Cheng-Xi
CX
Wei
State Key Laboratory of Medical Microbiology and Biosafety, Academy of Military Medical Sciences, Biotechnology Institute of Beijing, Beijing 100071, China. yue_junjie@126.com.
Jun-Jie
JJ
Yue
Tongliao Hospital, Tongliao 028000, China. zyf19991@126.com.
Yan-Fen
YF
Zhang
D001081:Apyrase / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012763:Shigella flexneri / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D014157:Transcription Factors; C058483:virG protein, Shigella flexneri; D001081:Apyrase