Kojima Takaaki, Mizoguchi Takuro, Ota Eri, Hata Jumpei, Homma Keisuke, Zhu Bo, Hitomi Kiyotaka, Nakano Hideo
Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Graduate School of Pharmaceutical Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
J Biosci Bioeng. 2016 Feb;121(2):147-53. doi: 10.1016/j.jbiosc.2015.06.003. Epub 2015 Oct 27.
A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities.
一种新型的DNA结合蛋白标签scCro-tag已被开发出来，它是噬菌体λ Cro阻遏蛋白的单链衍生物，可通过与被scCro蛋白紧密结合的OR共有DNA（ORC）结合，将目标蛋白（POI）固定在固体支持物上。scCro-tag通过支架DNA成功地将转谷氨酰胺酶2（TGase 2）底物和锰过氧化物酶（MnP）与微珠结合在一起。所得的蛋白质包被微珠可用于通过流式细胞术对酶活性进行功能分析。通过增加ORC的数量可以提高结合在微珠上的蛋白质的量。此外，使用无细胞蛋白质合成系统合成的带有scCro-tag的蛋白质也被固定在微珠上，这表明这种基于微珠的系统将适用于各种酶活性的高通量分析。
