Yang M, Wu Z, Fields S
Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook 11794, USA.
Nucleic Acids Res. 1995 Apr 11;23(7):1152-6. doi: 10.1093/nar/23.7.1152.
The yeast two-hybrid system was used to screen a library of random peptides fused to a transcriptional activation domain in order to identify peptides capable of binding to the retinoblastoma protein (Rb). Seven peptides were identified, all of which contain the Leu-X-Cys-X-Glu motif found in Rb-binding proteins, although their activity in the yeast assay varied over a 40-fold range. Mutagenesis of the DNA encoding two of these peptides followed by screening in the two-hybrid system allowed the delineation of residues apart from the invariant Leu, Cys and Glu that affect binding to Rb. Binding affinities of a peptide and one of its variants to Rb, determined by surface plasmon resonance, correlated with results from the two-hybrid assay. This method offers several advantageous features compared to existing technology for screening peptide libraries: in vivo detection of protein-peptide interactions, high sensitivity, the capacity for rapid genetic screening to identify stronger and weaker binding peptide variants, and the use of a simple assay (transcriptional activity) as a means to assess binding affinity.
酵母双杂交系统用于筛选与转录激活结构域融合的随机肽文库，以鉴定能够与视网膜母细胞瘤蛋白（Rb）结合的肽。共鉴定出7种肽，它们均含有在Rb结合蛋白中发现的Leu-X-Cys-X-Glu基序，尽管它们在酵母试验中的活性在40倍的范围内有所不同。对编码其中两种肽的DNA进行诱变，然后在双杂交系统中进行筛选，从而确定了除影响与Rb结合的不变Leu、Cys和Glu之外的其他残基。通过表面等离子体共振测定的一种肽及其变体之一与Rb的结合亲和力与双杂交试验的结果相关。与现有筛选肽文库的技术相比，该方法具有几个优点：体内检测蛋白质-肽相互作用、高灵敏度、能够通过快速遗传筛选鉴定结合更强和更弱的肽变体，以及使用简单的试验（转录活性）作为评估结合亲和力的手段。
