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    • 4. 发明申请
    • Method of Isolating P450 Gene
    • 分离P450基因的方法
    • US20080220419A1
    • 2008-09-11
    • US11665843
    • 2005-11-02
    • Mitsutoshi KubotaMiho NodateKenichi NodateTaku UchiyamaNorihiko Misawa
    • Mitsutoshi KubotaMiho NodateKenichi NodateTaku UchiyamaNorihiko Misawa
    • C12Q1/68C07H21/00C07H1/00C12N15/63C07K14/00C12N15/00C12N9/04C12N1/00C12P7/00
    • C12N9/0077C07K2319/00
    • The present invention provides a method for preparing a hybrid gene. The method includes a step of amplifying a P450 gene fragment contained in a sample using primers designed on the basis of regions of a plurality of P450 in which amino acid sequences are highly conserved and a step of preparing the hybrid gene using the amplified fragments and a known P450 gene. The method includes no culturing step or a step of normalizing extracted DNAs and is useful in isolating a P450 gene from various microbial resources.The present invention further provides a fused cytochrome P450 monooxygenase containing a peptide which is linked to the C-terminus of a P450 protein with a linker portion disposed therebetween and which has the same function as that of a reductase domain contained in a cytochrome P450 monooxygenase originating from Rhodococcus sp. strain NCIMB 9784. This enables the construction of a high-efficiency electron transfer system useful for various P450 proteins and also enables the production of an active P450 monooxygenase.
    • 本发明提供了一种制备杂交基因的方法。 该方法包括使用基于其中氨基酸序列高度保守的多个P450的区域设计的引物和使用扩增片段制备杂交基因的步骤来扩增样品中包含的P450基因片段的步骤,以及 已知的P450基因。 该方法不包括培养步骤或标准化提取的DNA的步骤,并且可用于从各种微生物资源中分离P450基因。 本发明还提供一种融合的细胞色素P450单加氧酶,其含有与P450蛋白的C末端连接的肽,其中连接部位置于其间,并且具有与源于细胞色素P450单加氧酶的还原酶结构域相同的功能 来自红球菌属 应用NCIMB 9784.这使得能够构建可用于各种P450蛋白质的高效电子转移系统,并且还能够生产活性P450单加氧酶。