Department of Virology, Max von Pettenkofer Institute, Ludwig Maximilians University Munich, Germany.
Hum Gene Ther. 2010 Sep;21(9):1104-18. doi: 10.1089/hum.2010.034.
The bacteriophage-derived ϕC31 integrase system represents an attractive tool for site-directed recombination in mammalian cells. Its integration reaction is based on recombination between the attachment site attB within an episomal substrate plasmid and either the bacteriophage-derived wild-type attachment site attP or pseudo-attP attachment sites (attP') present in the mammalian genome. In the present study we aimed at increasing the safety and efficiency of ϕC31 integrase-mediated recombination by mutating the DNA-binding domain located at the C terminus. Using an alanine mutagenesis approach, we generated 22 ϕC31 point mutants that were screened for activities in mammalian cells. Intramolecular excision assays based on recombination between attB and attP revealed five mutants with 2-fold enhanced excision activity. Importantly, we also identified mutants showing enhanced recombination activities between attB and three previously described attP' sites detected in the mammalian genome, indicating that there may be enhanced specificity for these hot spots. Several mutants showed, in mammalian cells, integration activities that increased in a cell line-dependent manner. The combination of beneficial mutations in addition to optimization of the integrase plasmid dose enhanced integration efficiencies up to 5.5-fold. We also identified three ϕC31 integrase mutants that were recombination defective in all applied assays, suggesting that these amino acid residues are essential for the functionality of ϕC31 integrase in mammalian cells. In summary, we identified critical amino acid residues within the ϕC31 DNA-binding domain. With respect to site-directed recombination and genome engineering these findings have important implications for improved ϕC31 protein design.
噬菌体衍生的 ϕC31 整合酶系统代表了一种在哺乳动物细胞中进行定点重组的有吸引力的工具。其整合反应基于附加在质体载体上的 attB 与噬菌体衍生的野生型附着位点 attP 或存在于哺乳动物基因组中的假附着位点 attP'（attP'）之间的重组。在本研究中，我们旨在通过突变位于 C 末端的 DNA 结合域来提高 ϕC31 整合酶介导的重组的安全性和效率。使用丙氨酸诱变方法，我们生成了 22 个 ϕC31 点突变体，并在哺乳动物细胞中筛选其活性。基于 attB 和 attP 之间重组的分子内切除测定揭示了五个具有 2 倍增强切除活性的突变体。重要的是，我们还鉴定了在哺乳动物基因组中检测到的三个先前描述的 attP'位点之间具有增强重组活性的突变体，表明它们可能对这些热点具有增强的特异性。几个突变体在哺乳动物细胞中显示出与细胞系依赖性方式增加的整合活性。有益突变的组合以及整合酶质粒剂量的优化将整合效率提高了 5.5 倍。我们还鉴定了三个在所有应用测定中均发生重组缺陷的 ϕC31 整合酶突变体，这表明这些氨基酸残基对于 ϕC31 整合酶在哺乳动物细胞中的功能至关重要。总之，我们确定了 ϕC31 DNA 结合域内的关键氨基酸残基。就定向重组和基因组工程而言，这些发现对改进 ϕC31 蛋白设计具有重要意义。
