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首页> 外文期刊>Journal of Molecular Biology >Directed evolution of protein inhibitors of DNA-nucleases by in vitro compartmentalization (IVC) and nano-droplet delivery.
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Directed evolution of protein inhibitors of DNA-nucleases by in vitro compartmentalization (IVC) and nano-droplet delivery.

机译：通过体外区室化（IVC）和纳米液滴递送指导DNA核酸酶蛋白抑制剂的进化。

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In vitro compartmentalization (IVC) uses water-in-oil emulsions to create artificial cell-like compartments in which genes can be individually transcribed and translated. Here, we present a new application of IVC for the selection of DNA-nuclease inhibitors. We developed a nano-droplets delivery system that allows the transport of various solutes, including metal ions, into the emulsion droplets. This transport mechanism was used to regulate the activity of colicin nucleases that were co-compartmentalized with the genes, so that the nucleases were activated by nickel or cobalt ions only after the potential inhibitor genes have been translated. Thus, genes encoding nuclease inhibitors survived the digestion and were subsequently amplified and isolated. Selection is therefore directly for inhibition, and not for binding of the nuclease. The stringency of selection can be easily modulated to give high enrichments (100-500-fold) and recoveries. We demonstrated its utility by selecting libraries of the geneencoding the cognate inhibitor of colicin E9 (immunity protein 9, or Im9) for inhibition of another colicin (ColE7). The in vitro evolved inhibitors show significant inhibition of ColE7 both in vitro and in vivo. These Im9 variants carry mutations into residues that determine the selectivity of the natural counterpart (Im7) while completely retaining the residues that are conserved throughout the family of immunity protein inhibitors. The in vitro evolution process confirms earlier hypotheses regarding the "dual recognition" binding mechanism and the way in which new colicin-immunity pairs diverged from existing ones.
机译：体外区室化（IVC）使用油包水乳状液来创建人造细胞样区室，在其中可以单独转录和翻译基因。在这里，我们介绍了IVC在DNA核酸酶抑制剂选择中的新应用。我们开发了一种纳米液滴输送系统，该系统可以将各种溶质（包括金属离子）输送到乳液液滴中。该转运机制用于调节与该基因共同分隔的大肠菌素核酸酶的活性，从而仅在潜在的抑制剂基因被翻译后，核酸酶才被镍或钴离子激活。因此，编码核酸酶抑制剂的基因在消化后幸存下来，随后被扩增和分离。因此，选择直接用于抑制，而不是用于核酸酶的结合。选择的严格性可以轻松调节，以实现高浓缩（100-500倍）和回收率。我们通过选择编码大肠菌素E9（免疫蛋白9或Im9）的同源抑制剂的基因文库来抑制另一种大肠菌素（ColE7），证明了其实用性。体外进化的抑制剂在体外和体内均显示出对ColE7的显着抑制。这些Im9变体将突变携带到残基中，这些残基确定了天然对应物（Im7）的选择性，同时完全保留了整个免疫蛋白抑制剂家族中保守的残基。体外进化过程证实了有关“双重识别”结合机制以及新的大肠菌素-免疫对与现有对的区别的早期假设。

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