三角酵母D-氨基酸氧化酶基因的克隆、测序及表达  

Cloning, Sequencing and Expression of D amino Acid Oxidase Gene

作　　者：刘洪波[1] 姜卫红[1] 杨蕴刘[1] 赵国屏[1] 焦瑞身[1] 

机构地区：[1]中国科学院上海植物生理研究所微生物次生代谢分子调控开放实验室

出　　处：《生物工程学报》1999年第3期337-337,共1页Chinese Journal of Biotechnology

基　　金："九五"国家攻关;上海市科学技术发展基金

摘　　要：利用跨越内含子的ＰＣＲ技术，从三角酵母（Ｔｒｉｇｏｎｏｐｓｉｓｖａｒｉａｂｉｌｉｓ）变种ＦＡ１１０中扩增得到Ｄ氨基酸氧化酶基因（ｄａａｏ），并通过ＴＡ克隆的方法将其克隆至ｐＧＥＭＴ载体。序列测定结果表明，所得ｄａａｏ基因的５′端内含子已被删除，基因总长度为１０７１ｂｐ，它与Ｔｒｉｇｏｎｏｐｓｉｓｖａｒｉａｂｉｌｉｓ的Ｄ氨基酸氧化酶同源性达９８３％，与Ｆｕｓａｒｉｕｍｓｏｌａｎｉ和Ｒｈｏｄｏｔｏｒｕｌａｇｒａｃｉｌｉｓ的同源性分别是３８９％和３０８％。为提高表达水平，又将此基因转移至高表达载体ｐＥＴ２８ｂ上，在大肠杆菌ＢＬ２１（ＤＥ３）中进行诱导表达。经ＩＰＴＧ诱导，目的蛋白的产生量可占菌体总蛋白量的４６％，分子量约为３８ｋＤ。Ｄ氨基酸氧化酶的活力可达８０２ｕ／Ｌ。The D amino acid oxidase gene( daao ) was cloned from the total DNA of Trigonopsis vairabilis by intron deleting PCR amplification and TA cloning methods. The daao gene consists of 1071bp encoding a protein of 357 amino acids. Comparing its nucleotide and amino acid sequences with DAAOs from other sources, considerable homologies were observed. The insertion of the daao gene into the high expression vector pET 28b allowed the expression of recombinant DAAO in E.coli as a soluble and catalytically active enzyme with a fermentation yield, in terms of DAAO units, of 802u/L. After IPTG induction target protein amounts to 46% of the total cell protein, its molecular weight is about 38 000 dalton.

关 键 词：三角酵母 氨基酸氧化酶 PCR 高表达 基因克隆 

分 类 号：Q949.326.1[生物学—植物学]