Cloning and characterization of novel GT-mismatch DNA binding protein

我们的目的是鉴定和鉴定一种新的GT错配结合蛋白nGTBP，它与高亲和力的TRTRNB(R；G或次黄嘌呤与T错配)结合(Takata-Yahiro，M等(2003)：Tohoku J.Exp.Med.)为了用LC/MS/MS测定nGTBP的部分氨基酸序列，初步建立了以下程序：30 kg超速离心HL60-c-15→Superose→肝素柱(2MKCl洗脱)→GC-匹配亲和柱(流经)→GT-错配亲和柱。然而，在SDS-PAGE中没有发现GT-错配探针的特异性蛋白条带。因此，我将我的实验转移到了nGTBP的分子克隆上。使用与CyBB的BP-183~-170相同的14聚体探针进行西南筛选，GT错配在-177处。然而，从3.6×10^7个克隆中没有获得确凿的阳性克隆，这表明有两种可能性：一是文库质量差，二是筛选条件平淡。后者通过在温和的KCl浓度下使用与BP-187~-168相同的3×20分子探针(GT-失配在-177)来克服。用该方法筛选出6×10~(-7)个以上的克隆，获得125个明显阳性克隆。然而，可重复的75个克隆中没有一个是针对GT不匹配的探针的。所有15个可重复克隆的插入序列都与转录蛋白YB-1的编码序列相同，这表明如果避免YB-1的非特异性结合，筛选过程是足够敏感的。这可能通过使用YB-1特异性DNA来完成。目前正在对人脑文库进行筛选。核抽提物的双向电泳法成功地适用于西南印迹，为LC/MS/MS测定nGTBP的部分氨基酸序列提供了可能。

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.