一种插入116个氨基酸的亮氨酰-tRNA合成酶具有高酶活力  

An Insertion Mutant of LeuRS with 116 Amino Acid Residues Has Full Activity

作　　者：黄颖[1] 凌晨[2] 李彤 童耕雷[1] 王恩多[2] 

机构地区：[1]上海交通大学生命科学技术学院,上海200240 [2]中国科学院上海生命科学研究院生物化学与细胞生物学研究所分子生物学国家重点实验室,上海200031

出　　处：《生物化学与生物物理学报》2003年第3期225-229,共5页

基　　金：国家高技术研究发展计划 ( 863计划 )项目 (No .2 0 0 1AA2 35 0 71);中国科学院创新项目 (No.KSCX2 2 0 4);上海市科学技术委员会项目 (No .0 2DJ14 0 5 67)资助~~

摘　　要：大肠杆菌亮氨酰 tRNA合成酶 (LeuRS)是第 1类氨基酰 tRNA合成酶 ,由 860个氨基酸残基组成 ,催化亮氨酸tRNA的亮氨酰化。研究发现 ,在它的CP1结构域内 3 68和 3 69间的肽键间插入 2 5 3～ 3 68的肽段 ,该插入变种的酶仍具有酶活力 ,取名为LeuRS C。由于这一插入变种的不稳定性 ,构建了His6 LeuRS C的表达质粒 ,用Ni NTA柱亲和层析的方法进行纯化。发现His6 LeuRS C虽然插入了 116个氨基酸残基 ,但仍具有全部的天然LeuRS的活力。测定了His6 LeuRS C的酶学动力学常数 。Escherichia coli leucyl-tRNA synthetase (LeuRS) belongs to calss I aminoacyl-tRNA synthetases. It consists of 860 amino acid residues and catalyzes the leucylation of tRNA leu. An insertion of its 253-368 peptide fragment between 368 to 369 in CP1 domain of this enzyme was shown to maintain the activity of the enzyme, and the insertion mutant was named as LeuRS-C. Because the insertion mutant of LeuRS was sensitive to operation of the purification, a plasmid containing the gene encoding LeuRS with His 6-tag at its N-terminus was constructed to facilitate the purification of His 6-LeuRS-C through one-step affinity chromatography on Ni-NTA column. The purified His 6-LeuRS-C had full activity as the native LeuRS with His-tag at the N-terminus (His 6-LeuRS), although the mutant enzyme had an insertion of 116 amino acid residues. The kinetic parameters of His 6-LeuRS-C were determined. The secondary structure estimated by CD spectrum and thermal stability of the insertion mutant was compared with those of His 6-LeuRS, respectively.

关 键 词：酶活力 大肠杆菌 亮氨酰-TRNA合成酶 插入变种 表达 纯化 

分 类 号：Q559[生物学—生物化学] Q753