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    Method for cloning and expression of SbfI restriction endonuclease and SbfI methylase in E. coli
    3.
    发明授权
    Method for cloning and expression of SbfI restriction endonuclease and SbfI methylase in E. coli 有权
    在大肠杆菌中克隆和表达SbfI限制性内切核酸酶和SbfI甲基化酶的方法

    公开(公告)号：US06958230B2

    公开(公告)日：2005-10-25

    申请号：US10668047

    申请日：2003-09-22

    申请人： Keith D. Lunnen ,  Theodore Davis ,  Geoffrey G. Wilson

    发明人： Keith D. Lunnen ,  Theodore Davis ,  Geoffrey G. Wilson

    IPC分类号： C12N1/21 ,  C12N9/10 ,  C12N9/22 ,  C12N15/55

    CPC分类号： C12N9/1007 ,  C12N9/22

    摘要： The present invention relates to: recombinant DNA encoding the SbfI restriction endonuclease as well as the SbfI methylase, and expression of the SbfI restriction endonuclease and SbfI methylase in E. coli cells containing the recombinant DNA; and methods for cloning the SbfI restriction gene (sbfIR) from Streptomyces species Bf-61 into E. coli by PCR. The method relied on primers based on DNA sequences predicted from amino acid sequences of the purified SbfI restriction endonuclease.

    摘要翻译： 本发明涉及编码SbfI限制性内切核酸酶以及SbfI甲基化酶的重组DNA，以及含有重组DNA的大肠杆菌细胞中SbfI限制性内切核酸酶和SbfI甲基化酶的表达。 以及通过PCR将来自Streptomyces物种Bf-61的SbfI限制性基因（sbfIR）克隆到大肠杆菌中的方法。 该方法依赖于基于纯化的SbfI限制性内切核酸酶的氨基酸序列预测的DNA序列的引物。

    Method for producing the AFL II restriction endonuclease and methylase
    4.
    发明授权
    Method for producing the AFL II restriction endonuclease and methylase 失效
    生产AFL II限制性内切核酸酶和甲基化酶的方法

    公开(公告)号：US5030569A

    公开(公告)日：1991-07-09

    申请号：US440438

    申请日：1989-11-20

    申请人： Keith D. Lunnen ,  Geoffrey G. Wilson

    发明人： Keith D. Lunnen ,  Geoffrey G. Wilson

    IPC分类号： C12N1/21 ,  C12N9/10 ,  C12N9/22

    CPC分类号： C12N9/22 ,  C12N9/1007

    摘要： The present invention is directed to a method for cloning and producing the Afl II restriction endonuclease by 1) introducing the restriction endonuclease gene from Anabaena flos-aquae into a host whereby the restriction gene is expressed; 2) fermenting the host which contains the plasmid encoding and expressing the Afl II restriction endonuclease activity, and 3) purifying the Afl II restriction endonuclease from the fermented host which contains the plasmid encoding and expressing the Afl II restriction endonuclease activity.

    摘要翻译： 本发明涉及一种用于克隆和产生AflⅡ限制性内切核酸酶的方法，其方法是1）将来自鱼腥藻的限制性内切核酸酶基因导入宿主，由此限制性基因被表达; 2）发酵含有编码并表达Af1Ⅱ限制性内切核酸酶活性的质粒的宿主，和3）从含有编码和表达AflⅡ限制性内切核酸酶活性的质粒的发酵宿主中纯化AflⅡ限制性内切核酸酶。

    Method for cloning and producing the SnaBI restriction endonuclease and purification of the recombinant SnaBI restriction endonuclease
    6.
    发明授权
    Method for cloning and producing the SnaBI restriction endonuclease and purification of the recombinant SnaBI restriction endonuclease 有权
    用于克隆和产生SnaBI限制性内切核酸酶并纯化重组SnaBI限制性内切核酸酶的方法

    公开(公告)号：US6025179A

    公开(公告)日：2000-02-15

    申请号：US143776

    申请日：1998-08-31

    申请人： Keith D. Lunnen ,  Huimin Kong ,  Geoffrey G Wilson

    发明人： Keith D. Lunnen ,  Huimin Kong ,  Geoffrey G Wilson

    IPC分类号： C12N15/09 ,  C12N1/15 ,  C12N1/19 ,  C12N1/21 ,  C12N5/10 ,  C12N9/10 ,  C12N9/16 ,  C12N9/22 ,  C12N15/52 ,  C12N15/55

    CPC分类号： C12N15/52 ,  C12N9/1007 ,  C12N9/22

    摘要： The methylase selection method was used to clone the SnaBI methylase gene (snaBIM) from Sphaerotilus natans (ATCC 15291). An active SnaBI methylase was cloned in E. coli using pSnaBI-2, a pUC19 derivative containing two SnaBI sites. Because methylase and restriction genes are usually located alongside each other in a restriction-modification systems, efforts were made to clone the downstream DNA by inverse PCR. Inverse PCR cloned the missing portion of the SnaBI endonuclease and also identified a control, or C, protein. The two open reading frames snaBIR (ORF1) and snaBIC (ORF2) converged toward the SnaBI methylase gene (ORF). Attempts to establish a snaBIR-recombinant plasmid expressing the SnaBI endonuclease in E.coli modified with SnaBI methylase failed. Overexpression of the SnaBI endonuclease in E. coli required the use of the heterospecific BsaAI methylase.

    摘要翻译： 甲基化酶选择方法用于从Sphaerotilus natans（ATCC 15291）克隆SnaBI甲基化酶基因（snaBIM）。 使用含有两个SnaBI位点的pUC19衍生物pSnaBI-2将活性SnaBI甲基化酶克隆到大肠杆菌中。 因为甲基化酶和限制性基因通常在限制性修饰系统中彼此并列，所以努力通过反向PCR克隆下游DNA。 反向PCR克隆了SnaBI内切核酸酶的缺失部分，并鉴定了对照或C蛋白。 snaBIR（ORF1）和snaBIC（ORF2）两个开放阅读框向SnaBI甲基化酶基因（ORF）收敛。 在SnaBI甲基化酶修饰的大肠杆菌中建立表达SnaBI内切核酸酶的snaBIR重组质粒的尝试失败。 大肠杆菌中SnaBI内切核酸酶的过表达需要使用异源特异性BsaAI甲基化酶。

    Method for producing the BGLI restriction endonuclease and methylase
    7.
    发明授权
    Method for producing the BGLI restriction endonuclease and methylase 失效
    生产BGLI限制性内切核酸酶和甲基化酶的方法

    公开(公告)号：US5366882A

    公开(公告)日：1994-11-22

    申请号：US169950

    申请日：1993-12-17

    申请人： Keith D. Lunnen ,  Geoffrey G. Wilson

    发明人： Keith D. Lunnen ,  Geoffrey G. Wilson

    IPC分类号： C12N9/10 ,  C12N9/22 ,  C12N15/53 ,  C12N15/70

    CPC分类号： C12N9/1007 ,  C12N9/22

    摘要： In accordance with the present invention there is provided an isolated DNA coding for the Bg/I restriction endonuclease and modification methylase derived from Bacillus globigii RUB561 stain, as well as related methods for cloning said recombinant DNA. The present invention also relates to clones which express recombinant Bg/I restriction endonuclease and recombinant modification methylase produced from Bg/I recombinant DNA and to methods for producing said enzymes.

    摘要翻译： 根据本发明，提供了编码Bg / I限制性内切核酸酶和衍生自芽孢杆菌RUB561染色体的修饰甲基化酶的分离的DNA，以及用于克隆所述重组DNA的相关方法。 本发明还涉及表达重组Bg / I限制性内切核酸酶和由Bg / I重组DNA产生的重组修饰甲基化酶的克隆和用于产生所述酶的方法。

    Method for cloning and producing the RsaI restriction endonuclease in E. coli and purification of the recombinant RsaI restriction endonuclease
    8.
    发明授权
    Method for cloning and producing the RsaI restriction endonuclease in E. coli and purification of the recombinant RsaI restriction endonuclease 有权
    在大肠杆菌中克隆和产生RsaI限制性内切核酸酶的方法，并重组RsaI限制性内切核酸酶的纯化

    公开(公告)号：US06210945B1

    公开(公告)日：2001-04-03

    申请号：US09587066

    申请日：2000-06-02

    申请人： Keith D. Lunnen ,  Richard D. Morgan ,  Timothy Meixsell ,  Geoffrey G. Wilson

    发明人： Keith D. Lunnen ,  Richard D. Morgan ,  Timothy Meixsell ,  Geoffrey G. Wilson

    IPC分类号： C12N922

    CPC分类号： C12N9/22

    摘要： RsaI, a restriction enzyme from the bacterium Rhodopseudomonas sphaeroides, recognizes the DNA sequence 5′-GTAC-3′. Because RsaI is commercially valuable, we sought to overproduce it by cloning the genes for RsaI and its accompanying, modification, enzyme. The ‘methylase-selection’ method, the customary procedure for cloning restriction and modification genes, was applied to RsaI. The method yielded clones containing the methylase gene (rsaIM), but none containing both the methylase gene and the restriction gene (rsaIR). Inverse-PCR was then used to recover sections of the DNA downstream of rsaIM. These sections were sequenced, and the sequences were joined in silico to reveal the gene organization of the RsaI R-M system. By comparing the coding potential of the DNA with the N-terminal amino acid sequence of the purified RsaI restriction enzyme, we discovered that the RsaI R and M genes, rather than being adjacent-the situation that pertains in most R-M systems-are separated by an intervening gene of unknown function. Based on this information, the rsaIR gene was cloned by PCR instead of methylase-selection. These new clones proved to be highly unstable, however, even in the presence of the rsaIM gene. Various attempts were made to stabilize the gene, but most met with failure. Stability was finally achieved by introducing a second methylase gene, mjaVM, to augment the protection provided by rsaIM, and by tightly controlling the expression of rsaIR using a special two-promoter, anti-sense transcription, expression vector.

    摘要翻译： 来自细菌Rhodopseudomonas sphaeroides的限制酶RsaI识别DNA序列5'-GTAC-3'。 由于RsaI具有商业价值，我们试图通过克隆RsaI及其伴随的修饰酶的基因来过度生产。 将“甲基化酶选择”方法，克隆限制和修饰基因的常规方法应用于RsaI，该方法产生含有甲基化酶基因（rsaIM）的克隆，但不含有甲基化酶基因和限制性基因（rsaIR）。 然后使用反向PCR来回收rsaIM下游DNA的切片，对这些切片进行测序，并将序列与硅胶连接以揭示RsaI RM系统的基因组织，通过将DNA的编码潜力与N 纯化的RsaI限制酶的末端氨基酸序列，我们发现RsaI R和M基因而不是与大多数RM系统相关的情况相邻 - 由未知功能的介入基因分离，基于该信息 ，通过PCR克隆rsaIR基因而不是甲基化酶选择，这些新克隆被证明是高度不稳定的，然而，即使在rsaIM基因的存在下，也进行了各种尝试 lize基因，但大多数遇到失败。 通过引入第二个甲基化酶基因mjaVM来增加rsaIM提供的保护，并通过使用特异的双启动子，反义转录，表达载体来严格控制rsaIR的表达，最终达到稳定性。