EspA is a novel fusion partner for expression of foreign proteins in Escherichia coli.
Escherichia coli secreted protein A (EspA) is a component of the type 3 secretion system (T3SS). The high level of expression when self-stimulated suggests that EspA may be used as a fusion partner. In the present study, EspA was used as a "fusion partner" to construct a fusion expression system, pEspA, in order to improve the expression and solubility of proteins from prokaryotes and eukaryotes. Target proteins were linked to the C-terminus of EspA by a linker containing a YAPQDP sequence, multiple cloning sites and an enterkinase cleavage site. Six proteins, IL-24, Stx2A1, Stx2B, S1, IntiminC300 and GFP, were expressed as EspA-fusion proteins using this vector. The expression level of each protein was enhanced by EspA and the majority of them (Stx2B, IntiminC300, GFP, Stx2A1, IL-24) were expressed in soluble form. EspA-fusion proteins can be purified by affinity chromatography (Sepharose chelated with EspA-specific monoclonal antibody) and by Ni(2+) affinity chromatography for they contain a 6× His tag at their C-terminus. In addition, IL-24 remains soluble and demonstrates certain anti-tumor activity after the removal of EspA by enterkinase. The EspA fusion expression system was efficient in enhancing expression levels and the solubility of target proteins.
Cheng
Yan
Y
Department of Clinical Microbiology and Immunology, College of Medical Laboratory, Third Military Medical University, Chongqing 400038, PR China.
Gu
Jiang
J
Wang
Hai-guang
HG
Yu
Shu
S
Liu
Yan-qing
YQ
Ning
Ya-lei
YL
Zou
Quan-ming
QM
Yu
Xue-jie
XJ
Mao
Xu-hu
XH
D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007378:Interleukins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism; D000099180:Interleukin-24
D004765:Enteropeptidase; D029968:Escherichia coli Proteins; D007378:Interleukins; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D000099180:Interleukin-24; C102051:EspA protein, E coli