Kim J S, Pabo C O
Howard Hughes Medical Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):2812-7. doi: 10.1073/pnas.95.6.2812.
Structure-based design was used to link zinc finger peptides and make poly-finger proteins that have dramatically enhanced affinity and specificity. Our studies focused on a fusion in which the three-finger Zif268 peptide was linked to a designed three-finger peptide (designated "NRE") that specifically recognizes a nuclear hormone response element. Gel shift assays indicate that this six-finger peptide, 268//NRE, binds to a composite 18-bp DNA site with a dissociation constant in the femtomolar range. We find that the slightly longer linkers used in this fusion protein provide a dramatic improvement in DNA-binding affinity, working much better than the canonical "TGEKP" linkers that have been used in previous studies. Tissue culture transfection experiments also show that the 268//NRE peptide is an extremely effective repressor, giving 72-fold repression when targeted to a binding site close to the transcription start site. Using this strategy, and linking peptides selected via phage display, should allow the design of novel DNA-binding proteins-with extraordinary affinity and specificity-for use in biological research and gene therapy.
基于结构的设计被用于连接锌指肽并构建具有显著增强的亲和力和特异性的多指蛋白。我们的研究集中在一种融合体上，其中三指的Zif268肽与一种专门识别核激素反应元件的设计三指肽（命名为“NRE”）相连。凝胶迁移实验表明，这种六指肽268//NRE与一个18碱基对的复合DNA位点结合，解离常数在飞摩尔范围内。我们发现，这种融合蛋白中使用的稍长的接头在DNA结合亲和力方面有显著提高，比先前研究中使用的典型“TGEKP”接头效果要好得多。组织培养转染实验还表明，268//NRE肽是一种极其有效的阻遏物，当靶向靠近转录起始位点的结合位点时，可产生72倍的阻遏作用。使用这种策略，并连接通过噬菌体展示选择的肽，应该能够设计出用于生物学研究和基因治疗的具有非凡亲和力和特异性的新型DNA结合蛋白。
