三角酵母D—氨基酸氧化酶基因的克隆,测序及表达

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。

Cloning, Sequencing and Expression of D amino Acid Oxidase Gene

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.