Yang Yong-Min, Barankiewicz Teresa J, He Mingyue, Taussig Michael J, Chen Swey-Shen
The Institute of Genetics, San Diego, CA 92121-2233, USA.
Biochem Biophys Res Commun. 2007 Jul 27;359(2):251-7. doi: 10.1016/j.bbrc.2007.05.083. Epub 2007 May 24.
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 (Ckappa) 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 Ckappa (3') were selected by anti-GFP or anti-Ckappa 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.
核糖体展示是一种无细胞系统，它通过遗传物质（mRNA）与其表型（蛋白质）产物的物理结合来实现基因筛选。虽然核糖体展示通常用于通过淘选固定化抗原从大型文库中筛选单链抗体，但我们已将核糖体展示调整为以“反向”形式使用，以便针对固相抗体筛选高亲和力抗原决定簇。为了构建一个抗原支架，将编码绿色荧光蛋白（GFP）的DNA与缺失终止密码子的轻链恒定结构域（Ckappa）融合，并带有5'信号（T7启动子、科扎克序列），从而能够在真核无细胞系统中进行耦合转录/翻译。分别通过与磁珠偶联的抗GFP或抗Ckappa抗体，筛选GFP（5'端）或Ckappa（3'端）上的表位。筛选后，通过原位PCR直接从蛋白质-核糖体-mRNA（PRM）复合物中扩增mRNA，随后进行内部扩增和重组PCR。与固相抗GFP抗体进行一轮相互作用后，仅10fg的1kb DNA构建体（即约7500个分子）就能被回收。该平台对抗原-抗体相互作用具有高度特异性和敏感性，可能允许对支架蛋白的高亲和力抗原变体进行筛选和改造。
