Development of chemical and rapid identification of the target molecules and ligand binding sites with high resolution

开发高分辨率的化学和快速鉴定靶分子和配体结合位点的方法

基本信息

批准号：
12470483
负责人：
NAKAYAMA Hitoshi
金额：
$ 8.64万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

In this project we aim to develop a method for rapid identification of the target molecules and ligand binding sites with high resolution in the recourse of immunoaffinity purification by anti-ligand antibodies, followed by moderen mass spectrometry. We planed to develop the method for identifying the binding site(s) of glutathione conjugate in cMOAT, an ATP-driven drug trasporter. During the passed 3 years, we obtained the results as follows. (1) We raised a antibody against hapten having analogous structure to photolabeling reagent of TOY-SG, a glutathione derivative. The antibody has a high titer to able to recognize hapten at sub-picomole level. (2) cMOAT protein that was photolabeled by TOY-SG was prepared and purified. We established the conditions of Lys-C digestion, which gave the photolabeled fragments of 3-4 kDa, proper size for MS nalysis. The photolabeled fragments were subjected to immunoaffinify purification using the anti-hapten antibody, but the yield was as low as 〜5%, which was not applicable for further MS analysis. (3) We fractionated the Lys-C fragments by HPLC as an alternative method and the fractions containing photolabeled fragments was analyzed by MALDI-TOF MS. We revealed two new photolabeled sites that located near nucleotide binding sites. The sites had not identified by any other methods such as site-directed mutagenesis. (4) Efforts to raise anti-hapten antibodies with much higher titer are still required.

在该项目中，我们旨在开发一种方法，以快速鉴定靶分子和配体结合位点，在通过抗体抗体恢复免疫植入纯化的过程中，其抗体抗体，然后进行现代质谱法。我们计划开发识别谷胱甘肽结合物在ATP驱动的药物载体CMOAT中的结合位点的方法。在过去的3年中，我们获得了如下的结果。（1）我们提出了一种针对与谷胱甘肽衍生物Toy-SG的光标试剂相似结构的抗体的抗体。该抗体具有很高的滴度，可以识别亚细胞水平的触觉。（2）制备并纯化由玩具-SG照相的CMOAT蛋白。我们确定了Lys-C消化的条件，该条件给出了3-4 kDa的光标记片段，适当的大小用于MS分析。使用抗Hapten抗体对光标记的片段进行免疫抗毒素的纯化，但产率高达约5％，不适用于进一步的MS分析。（3）我们通过HPLC作为替代方法将Lys-C片段分离，并通过Maldi-TOF MS分析了包含光标记片段的分数。我们揭示了位于核苷酸结合位点附近的两个新的光标记位点。这些站点尚未通过任何其他方法（例如定向诱变）识别。（4）仍然需要努力提高具有更高滴度的抗血统抗体。