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    Method for cloning and producing the SCaI restriction endonuclease in E. coli
    1.
    发明授权
    Method for cloning and producing the SCaI restriction endonuclease in E. coli 失效
    在大肠杆菌中克隆和产生SCaI限制性内切核酸酶的方法

    公开(公告)号：US5721126A

    公开(公告)日：1998-02-24

    申请号：US569806

    申请日：1995-12-08

    申请人： Shuang-Yong Xu ,  Jian-Ping Xiao

    发明人： Shuang-Yong Xu ,  Jian-Ping Xiao

    IPC分类号： C12N15/09 ,  C07H21/04 ,  C12N1/21 ,  C12N9/10 ,  C12N9/16 ,  C12N9/22 ,  C12N15/00 ,  C12N15/54 ,  C12N15/55 ,  C12R1/19 ,  C12R1/465 ,  C12N15/70

    CPC分类号： C12N9/22 ,  C12N9/1007

    摘要： The present invention relates to isolated DNA coding for the restriction endonuclease SCaI as well as to a method for cloning methylase genes from Streptomyces into E. coli by a modification of the methylase selection method. At first, the standard methylase gene selection method was tried to clone the SCaI methylase gene using a high-copy-number cloning vector pUC19 during library construction. The SCaI methylase gene was refractory to cloning by using pUC19, presumably due to the poor expression of the SCaI methylase gene in E. coli. If the SCaI methylase is not efficiently expressed in E. coli, the SCaI sites on the plasmid will not be sufficiently modified by the methylase. As a consequence, the plasmid will be cleaved and lost in the plasmid library after SCaI endonuclease challenge. Since the standard methylase selection did not work, the "endo-blue method" was tried to clone the SCaI endonuclease gene. Nineteen blue colonies were identified, but none of them yielded any detectable SCaI endonuclease activity. The SCaI endonuclease gene was first cloned by inverse PCR using primers that annealed to the end of the SCaI methylase gene. In order to increase the SCaI endonuclease expression in E. coli, an optimal ribosome binding site and spacing were engineered in front of the ATG start codon and the gene was inserted into expression vector pRRS.

    摘要翻译： 本发明涉及编码限制性内切核酸酶SCaI的分离的DNA以及通过甲基转移酶选择方法的修饰将来自链霉菌的甲基化酶基因克隆到大肠杆菌中的方法。 首先，在文库构建期间，使用高拷贝数克隆载体pUC19，尝试使用标准甲基化酶基因选择方法克隆SCaI甲基化酶基因。 SCaI甲基化酶基因通过使用pUC19进行克隆难以进行，大概是由于SCaI甲基化酶基因在大肠杆菌中的表达差。 如果SCaI甲基化酶不能在大肠杆菌中有效表达，则质粒上的SCaI位点将不会被甲基化酶充分修饰。 因此，在SCaI核酸内切酶攻击后，质粒将被切割并丢失在质粒文库中。 由于标准甲基化酶选择不起作用，因此试图克隆SCaI内切核酸酶基因的“内 - 蓝法”。 鉴定了十九个蓝色菌落，但没有一个产生任何可检测的SCaI核酸内切酶活性。 首先通过使用退火到SCaI甲基化酶基因末端的引物通过反向PCR克隆SCaI核酸内切酶基因。 为了增加大肠杆菌中的SCaI核酸内切酶表达，在ATG起始密码子前方设计了最佳的核糖体结合位点和间隔，并将该基因插入到表达载体pRRS中。

    Nicking Endonuclease Methods and Compositions
    2.
    发明申请
    Nicking Endonuclease Methods and Compositions 有权
    剔除核酸内切酶方法和组成

    公开(公告)号：US20080213860A1

    公开(公告)日：2008-09-04

    申请号：US11631438

    申请日：2005-07-22

    申请人： Shuang-Yong Xu ,  Zhenyu Zhu ,  Timothy Meixsell

    发明人： Shuang-Yong Xu ,  Zhenyu Zhu ,  Timothy Meixsell

    IPC分类号： C12N9/14

    CPC分类号： C12N9/22

    摘要： A nicking endonuclease is described which has an amino acid sequence with at least 70% identity to SEQ ID NO:6 and comprising a mutation at least one of an arginine or gutamic acid corresponding to position 507 and position 546 respectively in SEQ ID NO:6.

    摘要翻译： 描述了一种切口内切核酸酶，其具有与SEQ ID NO：6具有至少70％同一性的氨基酸序列，并且包含分别对应于SEQ ID NO：6中的位置507和位置546的精氨酸或谷氨酸中的至少一种的突变 。

    Method for cloning and expression of BsaI restriction endonuclease and BsaI methylase in E. coli
    7.
    发明授权
    Method for cloning and expression of BsaI restriction endonuclease and BsaI methylase in E. coli 有权
    在大肠杆菌中克隆和表达BsaI限制性内切核酸酶和BsaI甲基化酶的方法

    公开(公告)号：US06723546B2

    公开(公告)日：2004-04-20

    申请号：US10106275

    申请日：2002-03-26

    申请人： Zhenyu Zhu ,  Shuang-Yong Xu

    发明人： Zhenyu Zhu ,  Shuang-Yong Xu

    IPC分类号： C12N922

    CPC分类号： C12N9/1007 ,  C12N9/22

    摘要： The present invention relates to recombinant DNA which encodes the BsaI restriction endonuclease as well as BsaI methylase, expression of BsaI restriction endonuclease and BsaI methylase in E. coli cells containing the recombinant DNA, and purification of BsaI restriction endonuclease to near homogeneity.

    摘要翻译： 本发明涉及编码BsaI限制性内切核酸酶以及BsaI甲基化酶的重组DNA，BsaI限制性内切核酸酶和BsaI甲基化酶在含有重组DNA的大肠杆菌细胞中的表达，并将BsaI限制性内切核酸酶纯化至接近同质性。