Schaffitzel C, Hanes J, Jermutus L, Plückthun A
Biochemisches Institut, Universität Zürich, Wintherthurerstr. 190, Zürich, Switzerland.
J Immunol Methods. 1999 Dec 10;231(1-2):119-35. doi: 10.1016/s0022-1759(99)00149-0.
Combinatorial approaches in biology require appropriate screening methods for very large libraries. The library size, however, is almost always limited by the initial transformation steps following its assembly and ligation, as other all screening methods use cells or phages and viruses derived from them. Ribosome display is the first method for screening and selection of functional proteins performed completely in vitro and thus circumventing many drawbacks of in vivo systems. We review here the principle and applications of ribosome display for generating high-affinity antibodies from complex libraries. In ribosome display, the physical link between genotype and phenotype is accomplished by a mRNA-ribosome-protein complex that is used for selection. As this complex is stable for several days under appropriate conditions, very stringent selections can be performed. Ribosome display allows protein evolution through a built-in diversification of the initial library during selection cycles. Thus, the initial library size no longer limits the sequence space sampled. By this method, scFv fragments of antibodies with affinities in the low picomolar range have been obtained. As all steps of ribosome display are carried out entirely in vitro, reaction conditions of individual steps can be tailored to the requirements of the protein species investigated and the objectives of the selection or evolution experiment.
生物学中的组合方法需要针对非常大的文库采用合适的筛选方法。然而，文库大小几乎总是受到其组装和连接后初始转化步骤的限制，因为所有其他筛选方法都使用细胞或源自它们的噬菌体和病毒。核糖体展示是第一种完全在体外进行功能性蛋白质筛选和选择的方法，因此避免了体内系统的许多缺点。我们在此回顾核糖体展示从复杂文库中产生高亲和力抗体的原理和应用。在核糖体展示中，基因型和表型之间的物理联系是通过用于选择的mRNA-核糖体-蛋白质复合物实现的。由于这种复合物在适当条件下可稳定存在数天，因此可以进行非常严格的选择。核糖体展示允许通过在选择周期中对初始文库进行内置多样化来实现蛋白质进化。因此，初始文库大小不再限制所采样的序列空间。通过这种方法，已经获得了亲和力在低皮摩尔范围内的抗体单链可变片段。由于核糖体展示的所有步骤都完全在体外进行，因此各个步骤的反应条件可以根据所研究蛋白质种类的要求以及选择或进化实验的目标进行调整。
