EspA is a novel fusion partner for expression of foreign proteins in Escherichia coli

• Published in: Journal of biotechnology 2010-11, Vol.150 (3), p.380-388
• Main Authors: Cheng, Yan, Gu, Jiang, Wang, Hai-guang, Yu, Shu, Liu, Yan-qing, Ning, Ya-lei, Zou, Quan-ming, Yu, Xue-jie, Mao, Xu-hu
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Biotechnology; Cell Line; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Enteropeptidase - metabolism; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; EspA; Fundamental and applied biological sciences. Psychology; Fusion partner; Fusion protein; Humans; Interleukins - chemistry; Interleukins - genetics; Interleukins - isolation & purification; Interleukins - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Solubility; Thioredoxins - genetics; Thioredoxins - metabolism
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2010.09.940

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.