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    Endonuclease
    3.
    发明授权
    Endonuclease 失效
    核酸内切酶

    公开(公告)号：US06528296B1

    公开(公告)日：2003-03-04

    申请号：US09933700

    申请日：2001-08-20

    Applicant: Nobuhiro Morishima ,  Hikaru Mizumura ,  Takehiko Shibata

    Inventor： Nobuhiro Morishima ,  Hikaru Mizumura ,  Takehiko Shibata

    IPC: C12N922

    CPC classification number: C12N9/22

    Abstract: The present invention relates to a site-specific endonuclease which recognizes a specific nucleotide sequence, to a gene coding for the endonuclease, to a recombinant vector containing the gene, to a transformant containing the vector, and to a process for producing the endonuclease.

    Abstract translation: 本发明涉及将特异性核苷酸序列识别的位点特异性内切核酸酶，编码内切核酸酶的基因，含有该基因的重组载体，涉及含有该载体的转化体，以及生产该内切核酸酶的方法。

    Type II restriction endonuclease, HpyCH4III, obtainable from Helicobacter pylori CH4 and a process for producing the same
    5.
    发明授权
    Type II restriction endonuclease, HpyCH4III, obtainable from Helicobacter pylori CH4 and a process for producing the same 失效
    可从幽门螺杆菌CH4获得的新型II型限制性内切核酸酶HpyCH4III及其制备方法

    公开(公告)号：US06238904B1

    公开(公告)日：2001-05-29

    申请号：US09401870

    申请日：1999-09-23

    Applicant: Richard D. Morgan ,  Qing Xu

    Inventor： Richard D. Morgan ,  Qing Xu

    IPC: C12N922

    CPC classification number: C12N9/22

    Abstract: In accordance with the present invention, there is provided a novel restriction endonuclease and its DNA obtainable from Helicobacter pylori CH4 (NEB#1236), hereinafter referred to as “HpyCH4III”, which endonuclease: (1) recognizes the nucleotide sequence 5′-ACNGT-3′ in a double-stranded DNA molecule as shown below, 5′-ACN↓GT-3′ 3′-TG↑NCA-5′  (wherein G represents guanine, C represents cytosine, A represents adenine, T represents thymine and N represents either G, C, A, or T); (2) cleaves said sequence in the phosphodiester bonds between the N and G as indicated with the arrows; and (3) cleaves double-stranded PhiX174 DNA to produce 15 fragments, including fragments of 1284, 814, 536, 517, 454, 404, 302, 292, 270 and 222 base pairs, and 5 fragments smaller than 200 base pairs.

    Abstract translation: 根据本发明，提供了一种新型的限制性内切核酸酶及其可从幽门螺杆菌CH4（NEB＃1236）获得的DNA，以下称为“HpyCH4III”，该内切核酸酶：（1）识别核苷酸序列5'-ACNGT -3''，其中G表示鸟嘌呤，C表示胞嘧啶，A表示腺嘌呤，T表示胸腺嘧啶，T表示胸腺嘧啶核苷酸， N表示G，C，A或T）;（2）如箭头所示切割N和G之间的磷酸二酯键中的所述序列; （3）切割双链PhiX174 DNA以产生15个片段，包括1284,814,536,517,454,404,302,292,270和222个碱基对的片段，以及小于200个碱基对的5个片段。

    Method for cloning and producing the RsaI restriction endonuclease in E. coli and purification of the recombinant RsaI restriction endonuclease
    6.
    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

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

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

    IPC: C12N922

    CPC classification number: C12N9/22

    Abstract: 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.

    Abstract translation: 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。

    Mutant form of a cytotoxic ribonucleolytic protein which allows production by recombinant methods
    7.
    发明授权
    Mutant form of a cytotoxic ribonucleolytic protein which allows production by recombinant methods 失效
    突变形式的细胞毒性核糖核酸裂解蛋白，其允许通过重组方法生产

    公开(公告)号：US06649392B1

    公开(公告)日：2003-11-18

    申请号：US08626288

    申请日：1996-04-04

    Applicant: Richard J. Youle ,  Veena M. Vasandani ,  Yon-Neng Wu ,  Ester Boix ,  Wojciech Ardelt

    Inventor： Richard J. Youle ,  Veena M. Vasandani ,  Yon-Neng Wu ,  Ester Boix ,  Wojciech Ardelt

    IPC: C12N922

    CPC classification number: C12N9/22 ,  C07K14/463 ,  C12N2799/021

    Abstract: The present invention provides recombinant Onc (rOnc) compositions and methods. Recombinant Onc proteins of the invention have an amino terminal methionine and comprise an Onc polypeptide. The amino terminal methionine of the protein allows for recombinant production in a bacterial host cell. Cleaving the amino terminal methionine exposes the amino terminal glutamine of the polypeptide. The Onc polypeptide has an amino terminal glutamine. Cyclization of the amino terminal glutamine of the polypeptide to a pyroglutamyl residue provides rOnc polypeptides and proteins have anti-cancer and anti-viral activity.

    Abstract translation: 本发明提供重组Onc（rOn）组合物和方法。 本发明的重组Onc蛋白具有氨基末端甲硫氨酸并且包含Onc多肽。 蛋白质的氨基末端甲硫氨酸允许在细菌宿主细胞中重组生产。 切断氨基末端甲硫氨酸暴露多肽的氨基末端谷氨酰胺。 Onc多肽具有氨基末端谷氨酰胺。 将多肽的氨基末端谷氨酰胺环化成焦谷氨酰残基提供rOnc多肽和蛋白质具有抗癌和抗病毒活性。

    Human DNase
    9.
    发明授权
    Human DNase 失效
    人类DNase

    公开(公告)号：US06569660B1

    公开(公告)日：2003-05-27

    申请号：US09662746

    申请日：2000-09-15

    Applicant: Craig A. Rosen ,  Steven M. Ruben ,  Mark D. Adams

    Inventor： Craig A. Rosen ,  Steven M. Ruben ,  Mark D. Adams

    IPC: C12N922

    CPC classification number: C12N9/22 ,  A61K38/00

    Abstract: A human DNase polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for preventing and/or treating bronchopulmonary conditions. Diagnostic assays for identifying mutations in nucleic acid sequence encoding a polypeptide of the present invention and for detecting altered levels of the polypeptide of the present invention are also disclosed.

    Abstract translation: 公开了人DNase多肽和编码这种多肽的DNA（RNA），以及通过重组技术产生这种多肽的方法。 还公开了利用这种多肽来预防和/或治疗支气管肺疾病的方法。 还公开了用于鉴定编码本发明多肽的核酸序列中突变和用于检测本发明多肽的改变水平的诊断测定。

    Method for cloning and expression of Bpml restriction endonuclease in E. coli
    10.
    发明授权
    Method for cloning and expression of Bpml restriction endonuclease in E. coli 有权
    在大肠杆菌中克隆和表达Bpml限制性内切核酸酶的方法

    公开(公告)号：US06413758B1

    公开(公告)日：2002-07-02

    申请号：US09693146

    申请日：2000-10-20

    Applicant: Shuang-yong Xu ,  Jian-ping Xiao ,  Zhenyu Zhu

    Inventor： Shuang-yong Xu ,  Jian-ping Xiao ,  Zhenyu Zhu

    IPC: C12N922

    CPC classification number: C12N9/22 ,  C07K2319/00 ,  C12N9/1007

    Abstract: The present invention relates to recombinant DNA which encodes the BpmI restriction endonuclease as well as BpmI methyltransferase, expression of BpmI restriction endonuclease from E. coli cells containing the recombinant DNA. BpmI endonuclease is a fusion of two distinct elements with a possible structural domains of restriction-methylation-specificity (R-M-S). This domain organization is analogous to the type I restriction-modification system with three distinct subunits, restriction, methylation, and specificity (R, M, and S). Because BpmI is quite distinct to other type IIs restriction enzymes, it is proposed that BpmI belongs to a subgroup of type II restriction enzymes called type IIf (f stands for fusion of restriction-modification-specificity domains). The Type IIf group of restriction enzyme includes Eco57I, BpmI, GsuI, BseRI and some other restriction enzymes that cut downstream sequences at long distance, 10-20 bp downstream of recognition sequence, such as MmeI (N20/N18)).

    Abstract translation: 本发明涉及编码BpmI限制性内切核酸酶以及BpmI甲基转移酶的重组DNA，含有重组DNA的大肠杆菌细胞中BpmI限制性内切核酸酶的表达。 BpmI内切核酸酶是具有限制性甲基化特异性（R-M-S）的可能结构域的两个不同元件的融合物。 这种结构域组织类似于具有三个不同亚基，限制性，甲基化和特异性（R，M和S）的I型限制性修饰系统。 由于BpmI与其他II型限制性内切酶相当不同，所以提出BpmI属于称为IIf型的II型限制酶亚型（f代表限制性修饰特异性结构域的融合）。 IIf类限制酶包括Eco57I，BpmI，GsuI，BseRI和一些其他限制酶，其在识别序列下游10-20bp处长距离切割下游序列，例如MmeI（N20 / N18））。

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