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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Highly specific zinc finger proteins obtained by directed domain shuffling and cell-based selection.
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Highly specific zinc finger proteins obtained by directed domain shuffling and cell-based selection.

机译：通过定向域改组和基于细胞的选择获得高度特异性的锌指蛋白。

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摘要

Engineered Cys2His2 zinc finger proteins (ZFPs) can mediate regulation of endogenous gene expression in mammalian cells. Ideally, all zinc fingers in an engineered multifinger protein should be optimized concurrently because cooperative and context-dependent contacts can affect DNA recognition. However, the simultaneous selection of key contacts in even three fingers from fully randomized libraries would require the consideration of >1024 possible combinations. To address this challenge, we have developed a novel strategy that utilizes directed domain shuffling and rapid cell-based selections. Unlike previously described methods, our strategy is amenable to scale-up and does not sacrifice combinatorial diversity. Using this approach, we have successfully isolated multifinger proteins with improved in vitro and in vivo function. Our results demonstrate that both DNA binding affinity and specificity are important for cellular function and also provide a general approach for optimizing multidomain proteins.
机译：工程化的Cys2His2锌指蛋白（ZFP）可以介导哺乳动物细胞内源基因表达的调节。理想情况下，工程化的多指蛋白中的所有锌指均应同时进行优化，因为合作和上下文相关的接触会影响DNA识别。但是，要从完全随机化的库中同时选择甚至三个手指中的按键接触，都需要考虑> 1024种可能的组合。为了解决这一挑战，我们开发了一种利用定向域改组和基于细胞的快速选择的新颖策略。与先前描述的方法不同，我们的策略适合扩大规模，并且不会牺牲组合多样性。使用这种方法，我们成功地分离了具有改善的体外和体内功能的多指蛋白。我们的结果表明，DNA结合亲和力和特异性对于细胞功能都很重要，也为优化多域蛋白提供了一种通用方法。

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