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1. 发明申请

US20080268507A1 Recombinant Dna Nicking Endonuclease and Uses Thereof 审中-公开
标题翻译：重组Dna Nicking内切核酸酶及其用途
公开(公告)号：US20080268507A1
公开(公告)日：2008-10-30
申请号：US11666148
申请日：2005-10-21
申请人： Shuang-yong Xu , Zhenyu Zhu , Shi-hong Chan , Yan Xu
发明人： Shuang-yong Xu , Zhenyu Zhu , Shi-hong Chan , Yan Xu
IPC分类号： C12P19/34 , C07H21/04 , C12N9/16 , C12N9/10 , C12N15/00 , C12N1/21
CPC分类号： C12N9/22 , C12N9/1241
摘要： Recombinant nicking endonucleases and associated methylases have been obtained and sequenced and their specificity has been defined. A mutant form of the nicking endonuclease has been cloned where the mutation includes deletion of amino acid sequences at the C-terminal end of the protein. The nicking enzymes have been used for a number of purposes including: amplifying DNA from as few cells as can be found in a single bacterial colony in the presence of a strand displacing polymerase; and for removing genomic DNA in a biological preparation where it is deemed to be a contaminant.
摘要翻译：已经获得重组切口内切核酸酶和相关的甲基化酶并测序并确定了它们的特异性。已经克隆了缺口内切核酸酶的突变形式，其中突变包括在蛋白质C末端缺失氨基酸序列。切口酶已经被用于许多目的，包括：在链置换聚合酶存在下，从单个细菌菌落中可以发现的细胞中扩增DNA; 并且用于在被认为是污染物的生物制剂中去除基因组DNA。

2. 发明申请

US20050003420A1 Recycled mutagenesis of restriction endonuclease toward enhanced catalytic activity 审中-公开
标题翻译：限制性内切核酸酶的再循环诱变增强催化活性
公开(公告)号：US20050003420A1
公开(公告)日：2005-01-06
申请号：US10874527
申请日：2004-06-23
申请人： Shuang-yong Xu , Zhenyu Zhu , Paul Riggs , Pei-chung Hsieh
发明人： Shuang-yong Xu , Zhenyu Zhu , Paul Riggs , Pei-chung Hsieh
IPC分类号： C12N9/22 , C12Q1/68 , C07H21/04 , C12P21/04
CPC分类号： C12N9/22
摘要： A method is described for increasing the activity of restriction endonuclease mutants that have altered binding or cleavage activities. Restriction endonuclease variants can carry one or more amino acid substitutions that change substrate specificity and at the same time decrease the enzyme catalytic activity. A method is described for isolating derivatives of the endonuclease variants by subjecting them to additional rounds of mutagenesis and screening in a dinD::lacZ indicator strain, such that second-site mutations within the nucleotide coding sequence of the endonuclease are obtained that increased the enzyme specific activity.
摘要翻译：描述了增加具有改变的结合或切割活性的限制性内切核酸酶突变体的活性的方法。限制性内切核酸酶变体可以携带一个或多个改变底物特异性并同时降低酶催化活性的氨基酸取代。描述了通过在dinD :: lacZ指示菌株中进行额外的诱变和筛选来分离内切核酸酶变体的衍生物的方法，使得得到增加酶的核酸内切酶的核苷酸编码序列内的第二位点突变具体活动。

3. 发明申请

US20050136462A1 Method for engineering nicking enzymes 审中-公开
标题翻译：工程切口酶的方法
公开(公告)号：US20050136462A1
公开(公告)日：2005-06-23
申请号：US11013235
申请日：2004-12-15
申请人： Zhenyu Zhu , Shuang-yong Xu
发明人： Zhenyu Zhu , Shuang-yong Xu
IPC分类号： C12N9/00 , C12N9/22 , C12N15/10 , C12P21/00 , C12Q1/68 , C12N15/85
CPC分类号： C12N9/22 , C12N15/102
摘要： Methods are provided for identifying novel strand-specific nicking endonucleases by means of in vitro backcrosses of mutagenized restriction endonuclease genes with their wild-type counterpart and identifying the resulting nicking endonucleases by their cleavage activity and their strand specificity. Examples of nicking endonucleases identified by this method include Nt.Bsa I and Nb.BsaI, Nt.BsmAI and Nb.BsmAI and Nt.BsmBI.
摘要翻译：提供了通过诱变的限制性内切核酸酶基因与其野生型对应物的体外回交来鉴定新的链特异性切口内切核酸酶的方法，并通过其切割活性和它们的链特异性鉴定所得到的切口内切核酸酶。通过该方法鉴定的切口内切核酸酶的实例包括Nt.Bsa I和Nb.BsaI，Nt.BsmAI和Nb.BsmAI和Nt.BsmBI。

4. 发明授权

US06596524B2 Method for cloning and expression of BsmAi restriction endonuclease and BsmAI methylase in E. coli 失效
标题翻译：用于在大肠杆菌中克隆和表达BsmAi限制性内切核酸酶和BsmAI甲基化酶的方法
公开(公告)号：US06596524B2
公开(公告)日：2003-07-22
申请号：US09957005
申请日：2001-09-20
申请人： Zhenyu Zhu , Jing Zhou , Shuang-yong Xu
发明人： Zhenyu Zhu , Jing Zhou , Shuang-yong Xu
IPC分类号： C12N922
CPC分类号： C12N9/22 , C12N9/1007
摘要： The present invention relates to recombinant DNA which encodes the BsmAI restriction endonuclease as well as BsmAI methylase, expression of BsmAI restriction endonuclease and BsmAI methylase in E. coli cells containing the recombinant DNA, and purification of BsmAI endonuclease to near homogeneity.
摘要翻译：本发明涉及编码BsmAI限制性内切核酸酶以及BsmAI甲基化酶的重组DNA，含有重组DNA的大肠杆菌细胞中BsmAI限制性内切核酸酶和BsmAI甲基化酶的表达，以及将BsmAI内切核酸酶纯化至接近同质性。

5. 发明授权

US06335190B1 Method for cloning and producing the BsmI restriction endonuclease in E. coli 失效
标题翻译：用于在大肠杆菌中克隆和产生BsmI限制性内切核酸酶的方法
公开(公告)号：US06335190B1
公开(公告)日：2002-01-01
申请号：US09693147
申请日：2000-10-20
申请人： Jing Zhou , Zhenyu Zhu , Shuang-yong Xu
发明人： Jing Zhou , Zhenyu Zhu , Shuang-yong Xu
IPC分类号： C12N1555
CPC分类号： C12N9/22 , C12N9/1007
摘要： The present invention relates to recombinant DNA which encodes the BsmI restriction endonuclease as well as BsmI methyltransferases, expression of BsmI restriction endonuclease in E. coil cells containing the recombinant DNA by using a low copy number T7 expression vector pACYC-T7ter, and purification of BsmI restriction endonuclease by heat treatment and chromatography through heparin Sepharose column.
摘要翻译：本发明涉及编码BsmI限制性内切核酸酶以及BsmI甲基转移酶的重组DNA，通过使用低拷贝数T7表达载体pACYC-T7ter，含有重组DNA的大肠杆菌细胞中BsmI限制性内切核酸酶的表达，以及纯化BsmI 通过热处理和通过肝素琼脂糖凝胶柱色谱法进行限制性内切核酸酶。

6. 发明授权

US06919194B2 Method for cloning and expression of Tth111II restriction endonuclease-methylase in E. coli 失效
标题翻译：在大肠杆菌中克隆和表达Tth111II限制性内切核酸酶 - 甲基化酶的方法
公开(公告)号：US06919194B2
公开(公告)日：2005-07-19
申请号：US10338731
申请日：2003-01-08
申请人： Zhenyu Zhu , Derek Robinson , Jack Benner , Shuang-yong Xu
发明人： Zhenyu Zhu , Derek Robinson , Jack Benner , Shuang-yong Xu
IPC分类号： C12N9/10 , C12N9/22 , C12N15/55
CPC分类号： C12N9/22 , C07K2319/00 , C12N9/1007
摘要： The present invention relates to recombinant DNA which encodes the Tth111II restriction endonuclease-methylase fusion protein (Tth111IIRM), expression of Tth111II restriction endonuclease-methylase fusion protein in E. coli cells containing the recombinant DNA, and purification of Tth111II endonuclease-methylase fusion protein to near homogeneity.
摘要翻译：本发明涉及编码Tth111II限制性内切核酸酶 - 甲基化酶融合蛋白（Tth111IIRM）的重组DNA，含有重组DNA的大肠杆菌细胞中Tth111II限制性内切核酸酶 - 甲基化酶融合蛋白的表达，以及Tth111II内切核酸酶 - 甲基化酶融合蛋白的纯化接近同质性。

7. 发明授权

US06514737B1 Method for cloning and expression of AsiSI restriction endonuclease and AsiSI methylase in E. coli 有权
标题翻译：在大肠杆菌中克隆和表达AsiSI限制性内切核酸酶和AsiSI甲基化酶的方法
公开(公告)号：US06514737B1
公开(公告)日：2003-02-04
申请号：US09933313
申请日：2001-08-20
申请人： Zhenyu Zhu , Shuang-yong Xu
发明人： Zhenyu Zhu , Shuang-yong Xu
IPC分类号： C12N912
CPC分类号： C12N9/1007 , C12N9/22
摘要： The present invention relates to recombinant DNA which encodes the AsiSI restriction endonuclease as well as AsiSI methylase, expression of AsiSI restriction endonuclease and AsiSI methylase in E. coli cells containing the recombinant DNA.
摘要翻译：本发明涉及编码AsiSI限制性内切核酸酶以及AsiSI甲基化酶，AsiSI限制性内切核酸酶和AsiSI甲基化酶在含有重组DNA的大肠杆菌细胞中的表达的重组DNA。

8. 发明授权

US06413758B1 Method for cloning and expression of Bpml restriction endonuclease in E. coli 有权
标题翻译：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
申请人： Shuang-yong Xu , Jian-ping Xiao , Zhenyu Zhu
发明人： Shuang-yong Xu , Jian-ping Xiao , Zhenyu Zhu
IPC分类号： C12N922
CPC分类号： C12N9/22 , C07K2319/00 , C12N9/1007
摘要： 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)).
摘要翻译：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））。

9. 发明授权

US07943303B2 Method for engineering strand-specific nicking endonucleases from restriction endonucleases 有权
标题翻译：用于从限制性内切核酸酶技术设计链特异性切口内切核酸酶的方法
公开(公告)号：US07943303B2
公开(公告)日：2011-05-17
申请号：US11013260
申请日：2004-12-15
申请人： Shuang-yong Xu , James Samuelson
发明人： Shuang-yong Xu , James Samuelson
IPC分类号： C12Q1/00 , C12N15/00 , C12N15/09 , C12N15/11 , C12N15/52
CPC分类号： C12N9/22 , C12Q1/44
摘要： Methods are provided for engineering novel strand-specific nicking endonucleases by means of an in vivo enrichment of a plasmid library containing a randomly mutagenized restriction endonuclease gene. The plasmids contain adjacent to the gene a cleavable or nickable sequence for cleaving or nicking by the endonuclease product of the gene and a second recognition site for a second endonuclease. The plasmid library is used to transform unmodified host cells. Plasmids from the cultured transformed cells may be analyzed by an in vitro assay for nicking and the nicked plasmids pooled and used to transform host cells. The product is then pooled and the single-stranded specificity of the endonuclease is then determined. The product is either cloned after amplification or identified by use of a selectable marker.
摘要翻译：提供了通过体内富集含有随机诱变的限制性内切核酸酶基因的质粒文库来工程化新型链特异性切口内切核酸酶的方法。质粒与基因相邻，具有可切割或切割的序列，用于由该基因的内切核酸酶产物切割或切割，以及用于第二内切核酸酶的第二识别位点。质粒文库用于转化未修饰的宿主细胞。来自培养的转化细胞的质粒可以通过体外测定法进行分析，用于切口并合并切口的质粒并用于转化宿主细胞。然后将产物合并，然后测定内切核酸酶的单链特异性。产物在扩增后克隆或通过使用选择性标记进行鉴定。

10. 发明授权

US6133008A Method for cloning and producing the TfiI Restriction endonuclease in E. coli 失效
标题翻译：在大肠杆菌中克隆和产生TfiI限制性内切核酸酶的方法
公开(公告)号：US6133008A
公开(公告)日：2000-10-17
申请号：US306881
申请日：1999-05-07
申请人： Shuang-yong Xu , Pei-Chung Hsieh
发明人： Shuang-yong Xu , Pei-Chung Hsieh
IPC分类号： C12N9/22 , C12N15/55
CPC分类号： C12N9/22
摘要： A genomic DNA library of Thermus filiformis was constructed using pBR322 as a cloning vector. The methylase selection method was used to clone the TfiI methylase gene (tfiIM). A clone carrying an active TfiI methylase was identified. After sequencing the complete TfiI methylase gene and its downstream DNA sequence, a recombinase homolog was found. Because the methylase and its cognate endonuclease gene are located in proximity to each other in a particular restriction-modification system, efforts were made to clone the upstream DNA by inverse PCR. After two rounds of inverse PCR, one open reading frame (ORF1) was found upstream of the TfiI methylase gene. This ORF1, containing a Shine-Dalgarno sequence and a TATA box on the upstream side, was cloned and expressed, and TfiI endonuclease activity was detected in crude cell extracts. It is concluded that ORF1 encodes TfiI restriction endonuclease.
摘要翻译：使用pBR322作为克隆载体构建了丝状丝菌的基因组DNA文库。甲基化酶选择法用于克隆TfiI甲基化酶基因（tfiIM）。鉴定携带活性TfiI甲基化酶的克隆。测序完整的TfiI甲基化酶基因及其下游DNA序列后，发现重组酶同源物。因为甲基化酶及其同源核酸内切酶基因在特定的限制性修饰体系中位于彼此附近，所以努力通过反向PCR克隆上游DNA。在两轮反向PCR后，在TfiI甲基化酶基因的上游发现一个开放阅读框（ORF1）。在上游侧含有Shine-Dalgarno序列和TATA盒的ORF1被克隆并表达，并在粗细胞提取物中检测到TfiI核酸内切酶活性。得出结论，ORF1编码TfiI限制性内切核酸酶。

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