使用在<i>大肠杆菌中</i>表达的可光活化抗体Fc结合蛋白对抗体进行共价和定向表面固定<i></i>,Analytical Chemistry

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使用在大肠杆菌中表达的可光活化抗体Fc结合蛋白对抗体进行共价和定向表面固定
Analytical Chemistry ( IF 6.7 ) Pub Date : 2016-09-14 00:00:00 , DOI: 10.1021/acs.analchem.6b02071
Yeolin Lee ₁ , Jiyun Jeong ₁ , Gabi Lee ₁ , Jeong Hee Moon ₁ , Myung Kyu Lee ₁

Affiliation

具有蛋白G最小结构域的Fc特异性抗体结合蛋白（FcBP）被广泛用于将定向良好的抗体固定在固体表面上，但是与FcBPs的非共价结合的抗体在含有大量抗体的血清中不稳定。在这里，我们报告新型光活化的FcBPs与在大肠杆菌中表达的光甲硫氨酸（pMet），在紫外线照射下与抗体诱导Fc特异性光交联。不幸的是，pMet不支持天然大肠杆菌系统中的蛋白质表达，因此我们还开发了一种工程化的甲硫氨酰tRNA合成酶（MRS5m）。在甲硫氨酸营养缺陷型大肠杆菌中，MRS5m蛋白的共表达成功诱导了可光激活的FcBP过表达。细胞。可光活化的FcBP可以通过其N端半胱氨酸残基和6xHis标签轻松固定在珠子和载玻片上。光交联到可光激活的FcBP磁珠上的抗体可抵抗血清抗体介导的解离，并有效捕获人血清中的抗原。此外，使用该系统制备的光交联抗体阵列可通过夹心免疫测定法灵敏地检测人血清中的抗原。可光活化的FcBP将广泛应用于微流控芯片，载玻片和纳米珠的各种表面上的定向抗体固定，这是开发灵敏免疫传感器所必需的。

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Covalent and Oriented Surface Immobilization of Antibody Using Photoactivatable Antibody Fc-Binding Protein Expressed in Escherichia coli

Fc-specific antibody binding proteins (FcBPs) with the minimal domain of protein G are widely used for immobilization of well-oriented antibodies onto solid surfaces, but the noncovalently bound antibodies to FcBPs are unstable in sera containing large amounts of antibodies. Here we report novel photoactivatable FcBPs with photomethionine (pMet) expressed in E. coli, which induce Fc-specific photo-cross-linking with antibodies upon UV irradiation. Unfortunately, pMet did not support protein expression in the native E. coli system, and therefore we also developed an engineered methionyl tRNA synthetase (MRS5m). Coexpression of MRS5m proteins successfully induced photoactivatable FcBP overexpression in methionine-auxotroph E. coli cells. The photoactivatable FcBPs could be easily immobilized on beads and slides via their N-terminal cysteine residues and 6xHis tag. The antibodies photo-cross-linked onto the photoactivatable FcBP-beads were resistant from serum-antibody mediated dissociation and efficiently captured antigens in human sera. Furthermore, photo-cross-linked antibody arrays prepared using this system allowed sensitive detection of antigens in human sera by sandwich immunoassay. The photoactivatable FcBPs will be widely applicable for well-oriented antibody immobilization on various surfaces of microfluidic chips, glass slides, and nanobeads, which are required for development of sensitive immunosensors.

更新日期：2016-09-14

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