Expanding LAGLIDADG endonuclease scaffold diversity by rapidly surveying evolutionary sequence space
Expanding LAGLIDADG endonuclease scaffold diversity by rapidly surveying evolutionary sequence space
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DOI：
10.1093/nar/gkr1303
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发表时间：
2012-06-01
影响因子：
14.9
通讯作者：
Scharenberg, Andrew M.
中科院分区：
文献类型：
--
作者：
Jacoby, Kyle;Metzger, Michael;Scharenberg, Andrew M.
LAGLIDADG归巢内切酶（LHEs）是一类高度特异性的DNA内切酶，能够识别长度为20bp的靶序列，因此其潜在的学术、生物技术和临床应用引起了人们的强烈兴趣。目前，合理设计LHEs以切割所需靶点的方法受到少数高质量天然LHEs作为蛋白质工程支架的限制，其中许多不适合基因靶向应用。解决这些限制的一个策略是识别具有优越生物物理或催化性能的现有LHEs的密切同源物。为了验证这一概念，我们搜索了公共序列数据库，以确定与LHE I-AniI同源的假定的LHE开放阅读框，并使用酵母表面显示的DNA结合和切割试验来快速调查预测蛋白质的子集。这些蛋白表现出一系列的表面表达能力，也表现出局部改变的结合和裂解特异性，具有一系列的体内裂解活性。在这些酶中，I-HjeMI在体内表现出最大的活性，并且易于结晶，可以进行比较结构分析。综上所述，我们的研究结果表明，即使是高度同源的LHEs也为生物技术应用提供了一种容易获得的相关支架资源，这些支架显示出不同的生化特性。
LAGLIDADG homing endonucleases (LHEs) are a family of highly specific DNA endonucleases capable of recognizing target sequences similar to 20 bp in length, thus drawing intense interest for their potential academic, biotechnological and clinical applications. Methods for rational design of LHEs to cleave desired target sites are presently limited by a small number of high-quality native LHEs to serve as scaffolds for protein engineering-many are unsatisfactory for gene targeting applications. One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties. To test this concept, we searched public sequence databases to identify putative LHE open reading frames homologous to the LHE I-AniI and used a DNA binding and cleavage assay using yeast surface display to rapidly survey a subset of the predicted proteins. These proteins exhibited a range of capacities for surface expression and also displayed locally altered binding and cleavage specificities with a range of in vivo cleavage activities. Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis. Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications.