深黄被孢霉苹果酸酶基因的克隆和表达
发布时间：2018-08-05 19:42
【摘要】：为了克隆与表达深黄被孢霉(Mortierella isabellina)M6-22苹果酸酶基因,根据深黄被孢霉M6-22转录组测序结果,以其c DNA为模板PCR扩增苹果酸酶基因编码序列,经测序验证分析后将扩增片段连接到表达载体pET-32a(+)中构建重组表达质粒pET32a MIME2并进一步转化入大肠杆菌BL21中进行诱导表达,经镍柱亲和纯化目的蛋白和酶活分析确定其为苹果酸酶.PCR扩增得到全长为1 815 bp的cDNA序列,序列分析表明该序列具有一个编码604个氨基酸的开放阅读框,预测编码蛋白分子量(Mr)为66.4×10~3.预测的氨基酸序列与已报道的苹果酸酶同样具有保守的2个二核苷酸结合结构域和1个二价金属离子结合结构域,因此是一个新的苹果酸酶基因序列,命名为MIME2,Gen Bank序列号为KU097323.将该序列转化大肠杆菌BL21中进行诱导表达,SDS-PAGE电泳检测到1条约67×10~3的蛋白条带表达,经镍柱纯化和酶活分析表明所纯化蛋白能催化苹果酸脱氢生成丙酮酸,同时生成NADPH,具有苹果酸酶的特性,其酶活大小为177.46 U/mg.以上结果证明所克隆的cDNA序列MIME2为1个新的苹果酸酶基因,基因编码蛋白具有苹果酸酶活性,可为深入研究苹果酸酶的结构和功能关系以及进一步应用奠定基础.
[Abstract]:In order to clone and express (Mortierella isabellina) M6-22 malase gene, the coding sequence of Malinase gene was amplified by PCR using c DNA as template, according to the results of transcriptome sequencing of M6-22. After sequencing, the amplified fragment was ligated into the expression vector pET-32a () to construct the recombinant expression plasmid pET32a MIME2, which was further transformed into Escherichia coli BL21 for induction expression. A 1 815 BP cDNA sequence was amplified by nickel column affinity purification and enzyme activity analysis. The result showed that the sequence had an open reading frame encoding 604 amino acids. The predicted molecular weight of the encoded protein (Mr) was 66.4 脳 10 ~ (-3). The predicted amino acid sequence has two conserved dinucleotide binding domains and a divalent metal ion-binding domain as well as the reported malonidase, so it is a new malic enzyme gene sequence named MIME2G Bank sequence number KU097323. The purified protein was transformed into Escherichia coli BL21 by SDS-PAGE, and the protein band of 67 脳 10 ~ 3 was detected by SDS-PAGE. The purified protein could catalyze the dehydrogenation of malate to pyruvate by nickel column purification and enzyme activity analysis, and the results showed that the purified protein could catalyze the dehydrogenation of malic acid to pyruvate. At the same time, NADPH, which has the characteristic of malic enzyme, has the activity of 177.46 U / mg. These results suggest that the cloned cDNA sequence MIME2 is a new malonase gene, and the gene encoding protein has malonase activity, which may lay a foundation for further study on the structure and functional relationship of malic enzyme and its further application.
【作者单位】： 昆明理工大学生命科学与技术学院;
【基金】：国家自然科学基金项目(31160016,31660454) 云南省应用基础研究基金资助项目(KKSA201126005) 教育部回国人员科研启动基金项目(KKQA201226003)资助~~
【分类号】：Q936;Q78
[Abstract]:In order to clone and express (Mortierella isabellina) M6-22 malase gene, the coding sequence of Malinase gene was amplified by PCR using c DNA as template, according to the results of transcriptome sequencing of M6-22. After sequencing, the amplified fragment was ligated into the expression vector pET-32a () to construct the recombinant expression plasmid pET32a MIME2, which was further transformed into Escherichia coli BL21 for induction expression. A 1 815 BP cDNA sequence was amplified by nickel column affinity purification and enzyme activity analysis. The result showed that the sequence had an open reading frame encoding 604 amino acids. The predicted molecular weight of the encoded protein (Mr) was 66.4 脳 10 ~ (-3). The predicted amino acid sequence has two conserved dinucleotide binding domains and a divalent metal ion-binding domain as well as the reported malonidase, so it is a new malic enzyme gene sequence named MIME2G Bank sequence number KU097323. The purified protein was transformed into Escherichia coli BL21 by SDS-PAGE, and the protein band of 67 脳 10 ~ 3 was detected by SDS-PAGE. The purified protein could catalyze the dehydrogenation of malate to pyruvate by nickel column purification and enzyme activity analysis, and the results showed that the purified protein could catalyze the dehydrogenation of malic acid to pyruvate. At the same time, NADPH, which has the characteristic of malic enzyme, has the activity of 177.46 U / mg. These results suggest that the cloned cDNA sequence MIME2 is a new malonase gene, and the gene encoding protein has malonase activity, which may lay a foundation for further study on the structure and functional relationship of malic enzyme and its further application.
【作者单位】： 昆明理工大学生命科学与技术学院;
【基金】：国家自然科学基金项目(31160016,31660454) 云南省应用基础研究基金资助项目(KKSA201126005) 教育部回国人员科研启动基金项目(KKQA201226003)资助~~
【分类号】：Q936;Q78
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