Kugeratski Fernanda G, Batista Michel, Inoue Alexandre Haruo, Ramos Bruno Dias, Krieger Marco Aurelio, Marchini Fabricio K
Laboratório de Genômica Funcional, Instituto Carlos Chagas, Fundação Oswaldo Cruz, Curitiba, PR, BR.
Mem Inst Oswaldo Cruz. 2015 Aug;110(5):687-90. doi: 10.1590/0074-02760150074. Epub 2015 Jul 21.
The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzigenes and is freely available for scientific community.
对数千种克氏锥虫基因进行功能表征仍然是一项挑战。与高通量克隆策略兼容的反向遗传学方法可以提供应对这一挑战所需的工具。我们之前发表了pTcGW平台，它由基于Gateway®技术携带不同N端融合标签选项的质粒载体组成。在此，我们展示了pTcGW载体的改进版1.1，其特点是结构完全灵活，允许在单个克隆步骤中轻松定制载体的每个元件。此外，N端和C端融合都有新的标签选项可用于蛋白质复合物纯化。新创建的三个载体已成功用于确定四种克氏锥虫蛋白的细胞定位。pTcGW平台1.1版可用于多种分析，如蛋白质过表达、蛋白质-蛋白质相互作用鉴定和蛋白质定位。这个强大且通用的工具能够为克氏锥虫基因添加有价值的功能信息，并且可供科学界免费使用。
