Chemoselective DNA conjugation of proteins to study multienzyme clomplexes

蛋白质的化学选择性 DNA 缀合研究多酶复合物

• 批准号: 223269940
• 负责人: Professor Dr. Christof M. Niemeyer
• 金额: --
• 依托单位: Arbeitsgruppe Naturstoffchemie und -biochemie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/223269940?language=en
• 关键词: Chemoselective; DNA; conjugation; proteins; study

This project concerns the development of a generic platform to generate multfunctional protein constructs useful for interrogation of biological systems. The platform technology is based on the combination of chemoselective protein ligation methods and structural DNA nanotechnology. In particular, we will establish the chemoenzymatic ligation of recombinant proteins derived from multimodular Polyketide Synthases (PKS) with DNA or PNA oligonucleotides, which are derivatized with a short peptide tag, serving as the ligation substrate of bacterial transpeptidase Sortase. The resulting DNA-/PNA-tagged PKS modules will be assembled by Watson-Crick base pairing in homogeneous solution and, in particular, on the surface of two-dimensional nucleic acid scaffolds generated by the DNA origami technique. In a highly modular approach, supramolecular multienzyme complexes will be built, in which the spatial configuration of the PKS domains is controlled by the DNA scaffold with nanometer precision. On the one hand, this project will contribute to the detailed understanding of the spatial organization, conformational flexibility and other fundamental principles governing multienzyme complexes. On the other hand, the platform developed here will enable ready access to numerous spatially well-defined multifunctional protein complexes for mimicking, manipulation and analysis of natural occuring supramolecular assemblies involved in gene regulation, signal transduction, division, or other cellular function and traits.

该项目涉及开发一个通用平台，以产生用于生物系统询问的多功能蛋白质构建体。该平台技术基于化学选择性蛋白质连接方法和结构DNA纳米技术的组合。特别是，我们将建立来自多模块聚酮合酶（PKS）的重组蛋白与DNA或PNA寡核苷酸的化学酶促连接，所述DNA或PNA寡核苷酸用短肽标签衍生，用作细菌转肽酶分选酶的连接底物。所得的DNA-/PNA-标记的PKS模块将通过沃森-克里克碱基配对在均匀溶液中组装，特别是在通过DNA折纸技术产生的二维核酸支架的表面上。在高度模块化的方法中，将构建超分子多酶复合物，其中PKS结构域的空间构型由具有纳米精度的DNA支架控制。一方面，该项目将有助于详细了解多酶复合物的空间组织，构象灵活性和其他基本原则。另一方面，这里开发的平台将使许多空间上明确定义的多功能蛋白质复合物能够容易地用于模拟、操纵和分析涉及基因调控、信号转导、分裂或其他细胞功能和性状的天然存在的超分子组装体。