大肠杆菌色氨酸酶基因的克隆与表达

Molecular Cloning and Expression of Tryptophanase Gene of Escherichia coli

应用ＰＣＲ技术从Ｅ．ｃｏｌｉＪＭ１０５中扩增出长约１．４Ｋｂ的色氨酸酶基因，将其插入高表达载体ｐＥＴ３ａ的ＮｄｅＩ／ＢａｍＨＩ位点，转化Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３），构建高产色氨酸酶基因工程菌。ＳＤＳ－ＰＡＧＥ电泳和薄层扫描表明，工程菌色氨酸酶的表达量占细胞总可溶性蛋白的６９．８％。酶活测定结果表明，７株工程菌色氨酸酶的活力比宿主菌均有不同程度的提高，其中ＷＷ－１１号比宿主菌高１６倍。

kb DNA fragment was obtained from E. coli JM 105 by PCR. This fragment was inserted into NdeI/BamHI sites of pET3a. The recombinant plasmids were transformed into host strain E. coli BL 21(DE3). High level expression of tryptophanase genetic engineering strains were constructed. SDS PAGE and TLC were conducted, which proved that tryptophanase was 69.8% of all proteins of engineering strain. The results of enzyme assays showed that the activities of tryptophanase from seven genetic engineering strains were higher than those of host strain to different extent, and the enzyme activity of No.11 was of 16 times as high as that of the host.