Cloning and characterization of novel GT-mismatch DNA binding protein

新型 GT 错配 DNA 结合蛋白的克隆和表征

• 批准号: 16590248
• 负责人: NAKAMURA Michio
• 金额: $ 2.24万
• 依托单位: Nagasaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590248/
• 关键词: GT mismatch DNA; Specific DNA sequence; Affinity chromatography; DNA修復

We aimed to identify and characterize nGTBP, a novel GT-mismatch binding protein that specifically binds to TRTRNB(R ; G or hypoxanthine mispaired with T) with a high affinity (Takata-Yahiro, M et al.(2003) : Tohoku J.Exp.Med.).1. In order to determine a partial amino acid sequence of nGTBP by LC/MS/MS, the following procedure was tentatively established : 30Kg ultracentrifugation of nuclear extracts of HL60-c-15→Superose→heparin column ( 2 M KCl elution)→GC-match affinity column (flow through)→GT-mismatch affinity column. No protein bands specific to GT-mismatch probe were, however, found in SDS-PAGE. I, therefore, shifted my experiment to the molecular cloning of nGTBP.2. A South-western screening was established using a 14-mer probe identical to bp-183〜-170 of CYBB with a GT-mismatch at-177. No definitively positive clones were, however, obtained from 3.6x10^7 clones, suggesting two possibilities ; one is poor quality of the library, and the other dull screening condition. The latter was overcome by using the high concentration of a probe of 3 x 20 mer identical to bp-187〜-168 with a GT-mismatch at-177) under the mild KCl concentration.3. More than 6 x 10^7 clones were screened by using the method mentioned above, and were obtained 125 apparently positive clones. None of reproducible 75 clones was, however, specific to GT-mismatched probe.4. All insert sequences of 15 reproducible clones had identical to the coding sequence for YB-1, a transcriptional protein, suggesting the screening procedure is sufficiently sensitive if non-specific binding of YB-1 is avoided. It may be done by using YB-1 specific DNA. Human cerebral library is now being screened.5. Two-dimentional electrophoresis of the nuclear extract was successfully adapted to a South-western blotting, re-emerging the possibility of the partial amino acid sequence determination of nGTBP by LC/MS/MS.

我们旨在鉴定和表征nGTBP，这是一种新型的gt错配结合蛋白，它以高亲和力特异性结合TRTRNB（R； G或次黄嘌呤与T错配）（Takata-Yahiro， M等人（2003）：Tohoku J.Exp.Med.）。为了通过LC/MS/MS确定nGTBP的部分氨基酸序列，初步建立了以下流程：30Kg HL60-c-15核提取物超离心→Superose→肝素柱（2m KCl洗脱）→GC-match亲和柱（流式）→GT-mismatch亲和柱。然而，SDS-PAGE中未发现特异于gt错配探针的蛋白条带。因此，我将实验转向了nGTBP.2的分子克隆。使用一种与CYBB的bp-183 ~ -170相同的14-mer探针建立了西南筛选，该探针具有gt -不匹配的at-177。然而，从3.6x10^7个克隆中没有获得明确的阳性克隆，这表明有两种可能性；一是书库质量差，二是筛选条件沉闷。后者通过在轻度KCl浓度下使用与bp-187 ~ -168相同的3 × 20 mer （gt -不匹配at-177）的高浓度探针来克服。用上述方法筛选了6 × 10^7个克隆，得到125个明显阳性克隆。然而，可复制的75个克隆中没有一个对gt错配探针具有特异性。15个可复制克隆的所有插入序列都与转录蛋白YB-1的编码序列相同，表明如果避免了YB-1的非特异性结合，则筛选过程足够敏感。它可以通过使用YB-1特异性DNA来完成。人类大脑库正在被筛选。核提取物的二维电泳成功适应了西南印迹法，重新实现了LC/MS/MS法测定nGTBP部分氨基酸序列的可能性。