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STRUCTURAL STUDY OF INTERACTION BETWEEN c-MYC PROTEIN AND ITS TARGET DNA OLIGOMER BY NMR

核磁共振法研究 c-MYC 蛋白与其目标 DNA 寡聚物之间的相互作用

基本信息

批准号：
04452304
负责人：
UESUGI Seiichi
金额：
$ 3.14万
依托单位：
YOKOHAMA NATIONAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04452304/
关键词：
c-MYC NMR MAX c-myc タンパク質 DNA がん遺伝子構造遺伝子工学相互作用

项目摘要

(1) Attempts to establish of a system for production of c-Myc protein in E.coliDNA fragments encoding a part of c-Myc protein (342-439) were subcloned into two types of E.coli expression vectors (pRIT5 and pQE). However, no expression of the protein was demonstrated by SDS-PAGE and Western blotting by anti-c-Myc exon 3 serum. Then, we compared frequency of codon usage of E.coli and c-Myc. Both of Arg condons at 366 and 367 amino acid residue of c-Myc are AGG which is rarely found in E.coli. It is usually found that poor expression of recombinant protein is due to the continuous rare condons. So, we exchange the condons to relatively abundant ones to improve the expression. But, we failed to the expression of the protein.(2) Establishment of production system of Max protein and structural analysis of the protein.Full length of DNA fragment encoding Max (p22) protein was cloned into pQE E.coli expression vector. By using this plasmid, we succeeded to express the protein in E.coli. About 20mg of the purified protein was obtained from one litter culture of the E.coli. After the refolding the protein by stepwise reduction of urea, we carried out CD analysis. CD spectra showed highly alpha-helical nature of the protein. Also, addition of DNA oligomer induced drastic increase of the alpha-helix content.

(1)将编码部分c-Myc蛋白的大肠杆菌dna片段（342-439）亚克隆到两种大肠杆菌表达载体（pRIT5和pQE）中，试图建立一种生产c-Myc蛋白的系统。然而，SDS-PAGE和抗c- myc外显子3血清的Western blotting未发现该蛋白的表达。然后，我们比较了大肠杆菌和c-Myc的密码子使用频率。c-Myc的366和367个氨基酸残基上的Arg共聚物都是在大肠杆菌中很少发现的AGG。通常发现重组蛋白表达不佳是由于持续的罕见条件所致。因此，我们将安全套换成相对丰富的安全套来改善表达。但是，我们没有成功的表达该蛋白。(2) Max蛋白生产体系的建立及蛋白结构分析。将编码Max （p22）蛋白的全长DNA片段克隆到pQE大肠杆菌表达载体上。利用该质粒，我们成功地在大肠杆菌中表达了该蛋白。从一个大肠杆菌的窝培养中获得约20mg纯化蛋白。通过尿素的逐步还原将蛋白重新折叠后，我们进行CD分析。CD光谱显示该蛋白具有高度的α -螺旋性质。同时，DNA寡聚物的加入使α -螺旋含量急剧增加。