Takahara Mari, Wakabayashi Rie, Minamihata Kosuke, Goto Masahiro, Kamiya Noriho
Department of Applied Chemistry, Graduate School of Engineering and ‡Division of Biotechnology, Center for Future Chemistry, Kyushu University , 744 Motooka, Fukuoka 819-0395, Japan.
Bioconjug Chem. 2017 Dec 20;28(12):2954-2961. doi: 10.1021/acs.bioconjchem.7b00594. Epub 2017 Nov 20.
DNA-protein conjugates are promising biomolecules for use in areas ranging from therapeutics to analysis because of the dual functionalities of DNA and protein. Conjugation requires site-specific and efficient covalent bond formation without impairing the activity of both biomolecules. Herein, we have focused on the use of a microbial transglutaminase (MTG) that catalyzes the cross-linking reaction between a glutamine residue and a primary amine. In a model bioconjugation, a highly MTG-reactive Gln (Q)-donor peptide (FYPLQMRG, FQ) was fused to enhanced green fluorescent protein (FQ-EGFP) and a primary amine-clustered DNA aptamer was enzymatically synthesized as a novel acyl-acceptor substrate of MTG, whose combination leads to efficient and convenient preparation of DNA-protein conjugates with high purity. Dual functionality of the obtained DNA-EGFP conjugate was evaluated by discrimination of cancer cells via c-Met receptor recognition ability of the DNA aptamer. The DNA aptamer-EGFP conjugate only showed fluorescence toward cells with c-Met overexpression, indicating the retention of the biochemical properties of the DNA and EGFP in the conjugated form.
由于DNA和蛋白质具有双重功能，DNA-蛋白质缀合物是一类很有前景的生物分子，可用于从治疗到分析等多个领域。缀合需要在不损害两种生物分子活性的情况下形成位点特异性且高效的共价键。在此，我们专注于使用一种微生物转谷氨酰胺酶（MTG），它催化谷氨酰胺残基与伯胺之间的交联反应。在一个模型生物缀合中，将一个对MTG具有高反应性的Gln（Q）供体肽（FYPLQMRG，FQ）与增强型绿色荧光蛋白融合（FQ-EGFP），并酶促合成一种伯胺聚集的DNA适配体作为MTG的新型酰基受体底物，二者结合可高效便捷地制备高纯度的DNA-蛋白质缀合物。通过DNA适配体对c-Met受体的识别能力来区分癌细胞，从而评估所得DNA-EGFP缀合物的双重功能。DNA适配体-EGFP缀合物仅对c-Met过表达的细胞发出荧光，这表明DNA和EGFP在缀合形式下保留了其生化特性。
