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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.

在本项目中，我们的目标是在抗配体抗体免疫亲和纯化的基础上，开发一种高分辨率快速鉴定靶分子和配体结合位点的方法，然后采用现代质谱法。我们计划开发一种方法来识别谷胱甘肽缀合物在cMOAT （atp驱动的药物转运体）中的结合位点。在过去的三年中，我们取得了如下成果。(1)制备了一种结构类似于谷胱甘肽衍生物TOY-SG光标记试剂的半抗原抗体。该抗体具有高滴度，能够识别亚皮摩尔水平的半抗原。(2)制备并纯化了TOY-SG光标记的cMOAT蛋白。我们建立了lysc酶切的条件，得到了3-4 kDa的光标记片段，适合进行质谱分析。光标记的片段用抗半抗原抗体进行免疫亲和纯化，但产率低至~ 5%，不适合进一步的MS分析。(3)我们用高效液相色谱法（HPLC）对Lys-C片段进行分离，用MALDI-TOF ms对含有光标记片段的片段进行分析。我们发现了两个位于核苷酸结合位点附近的新光标记位点。这些位点未被任何其他方法（如位点定向诱变）鉴定。(4)仍需努力提高抗半抗原抗体的效价。