Hao Dongyun, Ohme-Takagi Masaru, Yamasaki Kazuhiko
National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba 305-8566, Japan.
FEBS Lett. 2003 Feb 11;536(1-3):151-6. doi: 10.1016/s0014-5793(03)00045-0.
A novel method is described which rapidly determines specificity of DNA-binding proteins using a surface plasmon resonance (SPR) sensor chip. An oligohistidine-tagged DNA-binding domain of a transcription factor, NtERF2, was immobilised via nitrilotriacetic acid ligands to a sensor chip with an attenuated degree of carboxymethylation. DNA molecules were selected from a pool of randomised oligomers through binding to the immobilised protein and amplified by PCR. After several cycles of selection, during which binding was monitored by SPR, DNA sequences containing a consensus sequence were determined. The time necessary for one cycle is approximately 50 min, which is shorter than existing methods.
描述了一种新方法，该方法使用表面等离子体共振（SPR）传感器芯片快速确定DNA结合蛋白的特异性。转录因子NtERF2的寡聚组氨酸标记的DNA结合结构域通过次氮基三乙酸配体固定在羧甲基化程度减弱的传感器芯片上。通过与固定化蛋白结合从随机寡聚物池中选择DNA分子，并通过PCR进行扩增。在几个选择循环中，通过SPR监测结合情况，之后确定包含共有序列的DNA序列。一个循环所需的时间约为50分钟，比现有方法更短。
