Improved properties of FLP recombinase evolved by cycling mutagenesis
Improved properties of FLP recombinase evolved by cycling mutagenesis
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DOI：
10.1038/nbt0798-657
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发表时间：
1998-07-01
影响因子：
46.9
通讯作者：
Stewart, AF
中科院分区：
文献类型：
--
作者：
Buchholz, F;Angrand, PO;Stewart, AF
位点特异性重组酶FLP和Cre在许多生物系统的基因组工程中很有用。对它们酶学性质的操控为提高它们在不同宿主生物中的适用性提供了一种手段。我们选择操控FLP重组酶的热不稳定性。在大肠杆菌中基于lacZ的重组分析被用于一种蛋白质进化策略的筛选，该策略依赖于易错PCR和DNA改组。通过提高温度和降低蛋白质表达所施加的严格性不断增加的循环，并结合在相同严格性下的重复筛选循环以富集适应性提高的克隆，鉴定出了改良的FLP重组酶。一个第八代克隆（称为FLPe）在大肠杆菌中、体外、人类293细胞和小鼠胚胎干细胞中都显示出改良的特性。
The site-specific recombinases FLP and Cre are useful for genomic engineering in many living systems. Manipulation of their enzymatic properties offers a means to improve their applicability in different host organisms. We chose to manipulate the thermolabilty of FLP recombinase. A lacZ-based recombination assay in Escherichia coli was used for selection in a protein evolution strategy that relied on error-prone PCR and DNA shuffling. Improved FLP recombinases were identified through cycles of increasing stringency imposed by both raising temperature and reducing protein expression, combined with repetitive cycles of screening at the same stringency to enrich for clones with improved fitness. An eighth generation clone (termed FLPe) showed improved properties in E. coli, in vitro, in human 293- and mouse ES-cells.