Selection of antigenic markers on a GFP-Ckappa fusion scaffold with high sensitivity by eukaryotic ribosome display.
Selection of antigenic markers on a GFP-Ckappa fusion scaffold with high sensitivity by eukaryotic ribosome display.
复制标题
通过真核核糖体展示高灵敏度地选择 GFP-Ckappa 融合支架上的抗原标记。
DOI：
10.1016/j.bbrc.2007.05.083
复制
发表时间：
2007
影响因子：
3.1
通讯作者：
Chen,Swey-Shen
中科院分区：
文献类型：
--
作者：
Yang,Yong-Min;Barankiewicz,TeresaJ;He,Mingyue;Taussig,MichaelJ;Chen,Swey-Shen
核糖体展示是一种无细胞系统，允许通过遗传物质（mRNA）及其表型（蛋白质）产物的物理结合进行基因选择。虽然经常用于选择单链抗体从大型库通过淘选对固定化抗原，我们已经适应核糖体展示中使用的“反向”格式，以选择高亲和力的抗原决定簇对固相抗体。为了产生抗原性支架，将编码绿色荧光蛋白（GFP）的DNA融合至终止密码子缺失的轻链恒定结构域（Cκ），并具有5′信号（T7启动子，Kozak），使得能够在真核无细胞系统中偶联转录/翻译。分别通过与磁珠偶联的抗GFP或抗C κ抗体选择GFP（5′）或Cκ（3′）上的表位。选择后，mRNA直接从蛋白质-核糖体-mRNA（PRM）复合物中通过原位PCR扩增，然后通过内部扩增和重组PCR扩增。在与固相抗GFP抗体的单轮相互作用后，可以回收少至10 fg的1 kb DNA构建体，即约7500个分子。该平台对抗原-抗体相互作用具有高度特异性和敏感性，并且可以允许选择和重塑支架蛋白的高亲和力抗原变体。
Ribosome display is a cell-free system permitting gene selection through the physical association of genetic material (mRNA) and its phenotypic (protein) product. While often used to select single-chain antibodies from large libraries by panning against immobilized antigens, we have adapted ribosome display for use in the ‘reverse’ format in order to select high affinity antigenic determinants against solid-phase antibody. To create an antigenic scaffold, DNA encoding green fluorescent protein (GFP) was fused to a light chain constant domain (Cκ) with stop codon deleted, and with 5′ signals (T7 promoter, Kozak) enabling coupled transcription/translation in a eukaryotic cell-free system. Epitopes on either GFP (5′) or Cκ (3′) were selected by anti-GFP or anti-Cκ antibodies, respectively, coupled to magnetic beads. After selection, mRNA was amplified directly from protein–ribosome–mRNA (PRM) complexes by in situ PCR followed by internal amplification and reassembly PCR. As little as 10fg of the 1kb DNA construct, i.e. approximately 7500 molecules, could be recovered following a single round of interaction with solid-phase anti-GFP antibody. This platform is highly specific and sensitive for the antigen–antibody interaction and may permit selection and reshaping of high affinity antigenic variants of scaffold proteins.