Werner Stefan, Marillonnet Sylvestre, Hause Gerd, Klimyuk Victor, Gleba Yuri
Icon Genetics and University of Halle, Biocenter, Weinbergweg 22, D-06120 Halle (Saale), Germany.
Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17678-83. doi: 10.1073/pnas.0608869103. Epub 2006 Nov 7.
Earlier attempts to express peptides longer than 20 aa on the surface of tobamoviruses such as tobacco mosaic virus have failed. Surprisingly, we found that a functional fragment of protein A (133 aa) can be displayed on the surface of a tobamovirus as a C-terminal fusion to the coat protein via a 15-aa linker. The macromolecular nature of these nanoparticles allowed the design of a simple protocol for purification of mAbs with a recovery yield of 50% and > 90% product purity. The extremely dense packing of protein A on the nanoparticles (> 2,100 copies per viral particle) results in an immunoadsorbent with a binding capacity of 2 g mAb per g. This characteristic, combined with the high level of expression of the nanoparticles (> 3 g adsorbent per kg of leaf biomass), provides a very inexpensive self-assembling matrix that could meet the criteria for a single-use industrial immunoadsorbent for antibody purification.
早期尝试在烟草花叶病毒等烟草花叶病毒表面表达长度超过20个氨基酸的肽均告失败。令人惊讶的是，我们发现蛋白A的一个功能片段（133个氨基酸）可以通过一个15个氨基酸的接头作为外壳蛋白的C端融合体展示在烟草花叶病毒表面。这些纳米颗粒的大分子性质使得能够设计出一种简单的单克隆抗体纯化方案，回收率为50%，产品纯度>90%。蛋白A在纳米颗粒上的极其密集堆积（每个病毒颗粒>2100个拷贝）产生了一种免疫吸附剂，其结合能力为每克2克单克隆抗体。这一特性与纳米颗粒的高表达水平（每千克叶片生物量>3克吸附剂）相结合，提供了一种非常廉价的自组装基质，可满足一次性工业抗体纯化免疫吸附剂的标准。
