Bioconjug Chem. 2013 Aug 21;24(8):1314-23. doi: 10.1021/bc300551j.
In this study, fluorescent proteins (FPs) were engineered to self-assemble into protein particles inside recombinant Escherichia coli while mediating the display of various protein functionalities such as maltose binding protein or IgG binding domains of Protein A or G, respectively. Escherichia coli produced functional FP particles of up to 30% of cellular dry weight. The use of respective FP particles displaying certain binding domains in diagnostics and as bioseparation resins was demonstrated by direct comparison to commercial offerings. It was demonstrated that variable extensions (AVTS, FHKP, LAVG, or TS) of the N-terminus of FPs (GFP, YFP, CFP, HcRed) in combination with large C-terminal extensions such as translational fusion of the polyester synthase from Ralstonia eutropha or an aldolase from Escherichia coli led to extensive intracellular self-assembly of strongly fluorescent fusion protein particles of oval shape (0.5×1 μm). The strong fluorescent label of these bioparticles in combination with covalent display of protein functions provides a molecular toolbox for the design of self-assembled microparticles suitable for antibody-capture or ligand binding based diagnostic assays as well as the high affinity purification of target compounds such as antibodies.
在这项研究中，荧光蛋白（FPs）被设计成在重组大肠杆菌中自我组装成蛋白颗粒，同时介导各种蛋白功能的展示，如麦芽糖结合蛋白或分别来自蛋白 A 或 G 的 IgG 结合结构域。大肠杆菌生产的具有功能的 FP 颗粒可达细胞干重的 30%。通过与商业产品的直接比较，展示了分别使用展示特定结合结构域的 FP 颗粒在诊断和作为生物分离树脂中的应用。研究表明，FPs（GFP、YFP、CFP、HcRed）N 端的可变延伸（AVTS、FHKP、LAVG 或 TS）与大型 C 端延伸（如 Ralstonia eutropha 的聚酯合酶或大肠杆菌的醛缩酶的翻译融合）结合，导致强荧光融合蛋白颗粒的广泛的细胞内自组装形成椭圆形（0.5×1μm）。这些生物颗粒的强荧光标记与蛋白功能的共价展示相结合，为设计适合抗体捕获或基于配体结合的诊断测定的自组装微颗粒以及目标化合物（如抗体）的高亲和力纯化提供了一个分子工具箱。
