Shimada Josui, Maruyama Tatsuo, Hosogi Takuya, Tominaga Jo, Kamiya Noriho, Goto Masahiro
Department of Applied Chemistry, Graduate School of Engineering, Center for Future Chemistry, Kyushu University, 744 Moto-oka, Fukuoka, 819-0395, Japan.
Biotechnol Lett. 2008 Nov;30(11):2001-6. doi: 10.1007/s10529-008-9784-4. Epub 2008 Jul 5.
We propose a novel method to prepare a DNA-protein conjugate using histidine-tag (His-tag) chemistry. Oligo-DNA was modified with nitrilotriacetate (NTA), which has high affinity to a His-tag on recombinant protein via the complexation of Ni(2+). Investigations using a microplate which displayed a complementary DNA-strand revealed that a NTA-modified DNA-protein conjugate was formed and immobilized in the presence of Ni(2+) on the microplate. We then adopted alkaline phosphatase (AP) as a model protein, and application of the DNA-AP conjugate was demonstrated in a thrombin aptamer-based detection system with a detection limit of approximately 10 nM.
我们提出了一种使用组氨酸标签（His-tag）化学方法制备DNA-蛋白质缀合物的新方法。寡聚DNA用次氮基三乙酸（NTA）进行修饰，通过Ni(2+)的络合作用，NTA对重组蛋白上的His-tag具有高亲和力。使用展示互补DNA链的微孔板进行的研究表明，在微孔板上Ni(2+)存在的情况下，形成并固定了NTA修饰的DNA-蛋白质缀合物。然后我们采用碱性磷酸酶（AP）作为模型蛋白，并在基于凝血酶适体的检测系统中证明了DNA-AP缀合物的应用，检测限约为10 nM。
