Department of Clinical Microbiology and Immunology, College of Medical Laboratory, Third Military Medical University, Chongqing 400038, PR China.
J Biotechnol. 2010 Nov;150(3):380-8. doi: 10.1016/j.jbiotec.2010.09.940. Epub 2010 Sep 18.
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.
大肠杆菌分泌蛋白 A（EspA）是 III 型分泌系统（T3SS）的组成部分。自身刺激时的高表达水平表明，EspA 可能被用作融合伴侣。在本研究中，EspA 被用作“融合伴侣”来构建融合表达系统 pEspA，以提高原核和真核生物蛋白质的表达和可溶性。靶蛋白通过含有 YAPQDP 序列、多个克隆位点和肠激酶切割位点的接头与 EspA 的 C 末端连接。使用该载体表达了六种蛋白质，即 IL-24、Stx2A1、Stx2B、S1、IntiminC300 和 GFP。每个蛋白质的表达水平都通过 EspA 得到增强，其中大多数（Stx2B、IntiminC300、GFP、Stx2A1、IL-24）以可溶性形式表达。EspA 融合蛋白可通过亲和层析（与 EspA 特异性单克隆抗体螯合的琼脂糖凝胶）和 Ni（2+）亲和层析进行纯化，因为它们在 C 末端含有 6×His 标签。此外，肠激酶去除 EspA 后，IL-24 仍保持可溶性并表现出一定的抗肿瘤活性。EspA 融合表达系统在提高靶蛋白的表达水平和可溶性方面非常有效。
