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Construction of an Effective System for Expression of Manganese Catalase Gene from a Thermophilic Bacterium

嗜热细菌锰过氧化氢酶基因表达的有效系统的构建

基本信息

批准号：
08650945
负责人：
NAGAI Jun
金额：
$ 1.41万
依托单位：
TOTTORI UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08650945/
关键词：
CATALASE MANGANESE THERMOSTABLE ENZYME THERMUS GENE EXPRESSION INCLUSION BODIES DENATURATION-RENATURATION 遺伝子解析

项目摘要

1. NUCLEOTIDE SEQUENCING OF A MANGANESE CATALASE GENEA novel manganese catalase gene from a thermophilic bacterium, Thermus sp.YS 8-13 isolated from a Japanese hot spring, was cloned and its nucleotide was determined. The 909-base open reading frame encoded 302 amino acids of deduced molecular weight 33,303. Within a 386-base long5'-flanking sequence, a SD-sequence and a promoter region analogous to that of E.coli were found. A 102-base 3'-flanking region contained a terminator-like palindrome sequence. The deduced amino acid sequence was 34% homologous overall to the sequence of Lactobacillus plantarum manganese catalase, but a significantly higher homology was found in certain regions which may form manganese ion containing active site.2. EXPRESSION OF MANGANESE CATALASE GENE IN E.COLIThe structural gene of manganese catalase was ligated into a high expression plasmid, pET system, and a resultant plasmid (pETMNCAT) was introduced into E.coli strain BL21. Strain BL21/pETMNCAT expressed significant amounts of a 36kDa foreign protein as inclusion bodies. As the N-terminus amino acid sequence of this expressed protein was identical to that of purified manganese catalase from Thermus sp.YS 8-13, solubilization and refolding of this enzyme protein was attempted. After solubilization of the inclusion body with 8M guanidium chloride, and removal of guanidium chloride by dialysis in the presence of manganese ion, about half of the expressed protein was obtained as a soluble and active manganese catalase. The molecular weights of the subunit and native enzyme, as well as the Vmax and K_M values were all identical to those of the authentic Thermus sp.YS 8-13 catalase.

1.从日本温泉中分离的嗜热菌Thermus sp.YS 8-13中克隆了一个新的锰过氧化氢酶基因，并测定了其核苷酸序列。909个碱基的开放阅读框编码302个氨基酸，推测的相对分子质量为33,303。在一个386碱基的长5‘侧翼序列中，发现了一个SD序列和一个与E.Coli相似的启动子区域。102个碱基的3‘侧翼区含有终止子样的回文序列。推导出的氨基酸序列与植物乳杆菌锰过氧化氢酶的氨基酸序列有34%的同源性，但在某些可能形成含有活性部位的锰离子的区域发现了更高的同源性。锰过氧化氢酶基因在大肠杆菌中的表达将锰过氧化氢酶结构基因连接到高效表达载体pET系统中，构建成重组表达载体pETMNCAT，并将其导入大肠杆菌BL21中。菌株BL21/pETMNCAT表达大量36 kDa的外源蛋白作为包涵体。由于该表达蛋白的N-末端氨基酸序列与Thermus sp.YS8-13纯化的锰过氧化氢酶完全相同，因此对该酶蛋白进行了增溶和复性。包涵体用8M氯化胍溶解，在锰离子存在下透析除去氯化胍，表达的蛋白中约有一半是可溶性的、有活性的锰过氧化氢酶。亚基和天然酶的相对分子质量以及Vmax和K_M值均与Thermus sp.YS8-13过氧化氢酶的同源性。