Directed evolution of the site specificity of Cre recombinase
Directed evolution of the site specificity of Cre recombinase
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DOI：
10.1073/pnas.022039799
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发表时间：
2002-04-02
影响因子：
11.1
通讯作者：
Schultz, PG
中科院分区：
文献类型：
--
作者：
Santoro, SW;Schultz, PG
来自噬菌体 P1 的 Cre 重组酶可识别 34 位重组位点 loxP，具有精细的序列特异性，并催化 DNA 的位点特异性插入、切除或重排。为了更好地了解蛋白质-DNA 识别的分子基础并生成具有改变的特异性的重组酶，我们开发了一种定向进化策略，可用于识别识别变体 loxP 位点的重组酶。为了被选择，通过定向随机诱变产生的 Cre 变体文库的成员必须在体内快速催化位于报告质粒上的两个变体 loxP 位点之间的重组。重组导致荧光蛋白表达模式发生改变，可以通过流式细胞术进行鉴定。荧光激活细胞分选可用于阳性筛选识别新 IoxP 位点的重组酶变体，或阴性筛选不能识别野生型 loxP 位点的变体。单独使用阳性筛选会导致重组位点特异性的放松，而阳性和阴性筛选的组合会导致特异性的转变。其中一种已鉴定的重组酶选择性地重组新的重组位点，并以与野生型 Cre 相同的速率运行。对由此产生的 Cre 变体的序列进行分析可以深入了解这些改变的特异性的演变。这个系统和其他系统应该有助于我们对蛋白质-DNA 识别的理解，并最终可能用于进化定制的重组酶，用于基因研究和失活。
Cre recombinase from bacteriophage P1 recognizes a 34-by recombination site, loxP, with exquisite sequence specificity and catalyzes the site-specific insertion, excision, or rearrangement of DNA. To better understand the molecular basis of protein-DNA recognition and generate recombinases with altered specificities, we have developed a directed evolution strategy that can be used to identify recombinases that recognize variant loxP sites. To be selected, members of a library of Cre variants produced by targeted random mutagenesis must rapidly catalyze recombination, in vivo, between two variant loxP sites that are located on a reporter plasmid. Recombination results in an altered pattern of fluorescent protein expression that can be identified by flow cytometry. Fluorescence-activated cell sorting can be used either to screen positively for recombinase variants that recognize a novel IoxP site, or negatively for variants that cannot recognize the wild-type loxP site. The use of positive screening alone resulted in a relaxation of recombination site specificity, whereas a combination of positive and negative screening resulted in a switching of specificity. One of the identified recombinases selectively recombines a novel recombination site and operates at a rate identical to that of wild-type Cre. Analysis of the sequences of the resulting Cre variants provides insight into the evolution of these altered specificities. This and other systems should contribute to our understanding of protein-DNA recognition and may eventually be used to evolve custom-tailored recombinases that can be used for gene study and inactivation.