[Construction of prokaryotic expression vectors for tandem affinity purification].
OBJECTIVE
To construct prokaryotic expression vectors suitable for tandem affinity purification to study protein-protein interactions in bacteria.


METHODS
Two tandem affinity tag sequences, including the coding sequences of Protein G and streptavidin binding protein (SBP), as the N- and C- terminus of fusion proteins were designed and de novo synthesized. Constitutive expression vectors pNTAP and pCTAP were constructed using pUC18 as the backbone deleted of the lacI gene.


RESULTS
Two expression vectors pNTAP and pCTAP were successfully constructed, pNTAP showed substantial expression of the built-in tag protein GFPuv not only in Escherichia coli BL21 (DE3) but also in enterohemorrhagic Escherichia coli O157:H7 and Shigella flexneri 5a.


CONCLUSIONS
Of the two recombinant expression vectors successfully constructed, pNTAP can express the model protein for tandem affinity purification and could be used for studies of protein-protein interactions in some gram-negative pathogenic bacteria such as Escherichia coli and Shigella flexneri.
College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China. weiboweibo000000@163.com
Bo
B
Wei
Xiang
X
Liao
Wei
W
Zhou
Yuan
Y
Gao
Yu
Y
Wang
Jinbao
J
Ran
Long
L
Liang
Junjie
J
Yue
Bateer
B
Huhe
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011387:Prokaryotic Cells / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D011993:Recombinant Fusion Proteins