Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces (IBG 1), Hermann-von-Helmholtz-Platz, 76344 Eggenstein-Leopoldshafen (Germany).
Angew Chem Int Ed Engl. 2015 Jun 1;54(23):6745-50. doi: 10.1002/anie.201500175. Epub 2015 Apr 27.
The positioning of enzymes on DNA nanostructures for the study of spatial effects in interacting biomolecular assemblies requires chemically mild immobilization procedures as well as efficient means for separating unbound proteins from the assembled constructs. We herein report the exploitation of free-flow electrophoresis (FFE) for the purification of DNA origami structures decorated with biotechnologically relevant recombinant enzymes: the S-selective NADP(+)/NADPH-dependent oxidoreductase Gre2 from S. Cerevisiae and the reductase domain of the monooxygenase P450 BM3 from B. megaterium. The enzymes were fused with orthogonal tags to facilitate site-selective immobilization. FFE purification yielded enzyme-origami constructs whose specific activity was quantitatively analyzed. All origami-tethered enzymes were significantly more active than the free enzymes, thereby suggesting a protective influence of the large, highly charged DNA nanostructure on the stability of the proteins.
为了研究相互作用的生物分子组装体中的空间效应，需要将酶定位在 DNA 纳米结构上，这就需要化学性质温和的固定化程序以及从组装结构中有效分离未结合的蛋白质的方法。本文报道了利用自由流动电泳（FFE）纯化 DNA 折纸结构的方法，该结构用生物技术相关的重组酶进行了修饰：来自 S. cerevisiae 的 S 选择性 NADP（+）/NADPH 依赖性氧化还原酶 Gre2 和来自 B. megaterium 的单加氧酶 P450 BM3 的还原酶结构域。这些酶与正交标签融合，以方便进行位点选择性固定化。FFE 纯化得到的酶-折纸结构的比活性进行了定量分析。所有的折纸固定化酶的活性都明显高于游离酶，这表明大型、高电荷的 DNA 纳米结构对蛋白质的稳定性具有保护作用。
