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    Method for engineering strand-specific nicking endonucleases from restriction endonucleases
    4.
    发明授权
    Method for engineering strand-specific nicking endonucleases from restriction endonucleases 有权
    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。

    Method for cloning and producing the TfiI Restriction endonuclease in E. coli
    5.
    发明授权
    Method for cloning and producing the TfiI Restriction endonuclease in E. coli 失效
    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。

    Method for cloning and expression of BsrFI restriction endonuclease in E. coli
    6.
    发明授权
    Method for cloning and expression of BsrFI restriction endonuclease in E. coli 有权
    在大肠杆菌中克隆和表达BsrFI限制性内切核酸酶的方法

    公开(公告)号：US6066487A

    公开(公告)日：2000-05-23

    申请号：US307621

    申请日：1999-05-07

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

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

    IPC分类号： C12N9/22 ,  C12N15/55

    CPC分类号： C12N9/22

    摘要： BsrFI restriction enzyme was purified from Bacillus stearothermophilus to near homogeneity. The protein was sequenced to obtain its N-terminus amino acid sequence. A set of denegerate primers were synthesized based on the aa sequence. The first 18 codons encoding BsrFI restriction endonuclease (bsrFIR) was amplified by PCR and its coding sequence was obtained. The methylase selection method was used to clone BsrFI methylase gene (bsrFIM). Two clones were found to be resistant to BsrFI digstion. The entire insert in one clone was sequenced and the insert encodes the BsrFI methylase (M. BsrFI). In addition, a small truncated open reading frame adjacent to the methylase gene has homology to Cfr10I restriciton endonuclease in a BlastX homology search in Genbank database. BsrFI and Cfr10I are isoschizomer that recognizes and cleaves 5'R CCGGY3'. Two primers were used to amplify the bsrFIR gene, The forward primer is a degenerate primer designed from the N-terminus aa sequence and the reverse primer is the bona fide sequence derived from the BsrFI methylase.sup.+ clone. The bsrFIR gene was amplified by PCR, ligated into a T7 expression vector pET21at and the ligated DNA was transformed into premodified cells ER2566 [pLG339-BsrFIM]. The final expression strain is ER2566 [pLG339-BsrFIM, pET21at-BsrFIR]. Recombinant BsrFI activity was detected in E. coli cell extract. BsrFI is cloned from a thermophile Bacillus stearothermophilus. Thus, BsrFI a thermostable enzyme and it is active at 37.degree. C. to 65.degree. C.

    摘要翻译： 将BsrFI限制性酶从嗜热脂肪芽孢杆菌纯化至接近同质性。 对蛋白进行测序以获得其N-末端氨基酸序列。 基于aa序列合成了一组脱壳引物。 通过PCR扩增编码BsrFI限制性内切核酸酶（bsrFIR）的前18个密码子，获得编码序列。 甲基化酶选择法用于克隆BsrFI甲基化酶基因（bsrFIM）。 发现两个克隆对BsrFI突变具有抗性。 对一个克隆中的整个插入片段进行测序，插入序列编码BsrFI甲基化酶（M.BsrFI）。 此外，在Genbank数据库中的BlastX同源性搜索中，与甲基化酶基因相邻的小截断的开放阅读框与Cfr10I限制性内切核酸酶具有同源性。 BsrFI和Cfr10I是识别并切割5'R + E，cir + EE CCGGY3'的异构体。 使用两个引物来扩增bsrFIR基因。正向引物是从N末端aa序列设计的简并引物，反向引物是来自BsrFI甲基化酶+克隆的真正序列。 通过PCR扩增bsrFIR基因，连接到T7表达载体pET21at中，将连接的DNA转化到预变性细胞ER2566 [pLG339-BsrFIM]中。 最终的表达菌株是ER2566 [pLG339-BsrFIM，pET21at-BsrFIR]。 在大肠杆菌细胞提取物中检测到重组BsrFI活性。 从嗜热芽孢杆菌嗜热脂肪芽孢杆菌克隆BsrFI。 因此，BsrFI是一种热稳定酶，在37℃至65℃下是有活性的。

    Recombinant Dna Nicking Endonuclease and Uses Thereof
    7.
    发明申请
    Recombinant Dna Nicking Endonuclease and Uses Thereof 审中-公开
    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。

    Alteration of restriction endonuclease specificity by genetic selection
    8.
    发明授权
    Alteration of restriction endonuclease specificity by genetic selection 有权
    通过遗传选择改变限制性内切核酸酶特异性

    公开(公告)号：US07052897B2

    公开(公告)日：2006-05-30

    申请号：US10501196

    申请日：2003-01-09

    申请人： James C. Samuelson ,  Shuang-yong Xu

    发明人： James C. Samuelson ,  Shuang-yong Xu

    IPC分类号： C12N9/22 ,  C12N9/10

    CPC分类号： C12N9/16 ,  C12N9/22 ,  C12N15/102

    摘要： Methods and compositions are provided for altering the DNA recognition and cleavage characteristics of an endonuclease without prior knowledge of the endonuclease's three-dimensional structure and/or amino acid residues responsible for activity and/or specificity. Methods include subjecting a mutagenized endonuclease gene library to a genetic selection in prokaryotic cells which tolerate the expression of mutated endonuclease and where the endonuclease is active and determining the altered recognition-site specificity for the endonuclease.

    摘要翻译： 提供了方法和组合物，用于改变核酸内切酶的DNA识别和切割特征，而无需事先知道负责活性和/或特异性的内切核酸酶的三维结构和/或氨基酸残基。 方法包括将诱变的内切核酸酶基因文库进行基因选择，其中原核细胞可耐受突变的核酸内切酶的表达，并且其中内切核酸酶是活性的并且确定内切核酸酶的识别位点特异性的改变。

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

    公开(公告)号：US06387681B1

    公开(公告)日：2002-05-14

    申请号：US09428747

    申请日：1999-10-28

    申请人： Shuang-yong Xu ,  Jian-ping Xiao

    发明人： Shuang-yong Xu ,  Jian-ping Xiao

    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 NheI restriction endonuclease as well as NheI methyltransferase, expression of NheI restriction endonuclease from E. coli cells containing the recombinant DNA. An internal NdeI site in the nheIR gene was eliminated by a silent mutation. A new NdeI site was engineered at the start codon of nheIR gene. An NdeI-BamHI fragment containing nheIR gene was cloned into a T7 expression vector pAII17 and expressed in a premodified host ER2566 [pACYC-NheIM, pAII17-NheIR2]. The recombinant clone produces approximately 10 million units of NheI per gram of wet cells.

    摘要翻译： 本发明涉及编码NheI限制性内切核酸酶以及NheI甲基转移酶，含有重组DNA的大肠杆菌细胞中NheI限制性内切核酸酶表达的重组DNA。 nheIR基因内部的NdeI位点被沉默突变消除。 在nheIR基因的起始密码子处设计了一个新的NdeI位点。 将含有nheIR基因的NdeI-BamHI片段克隆到T7表达载体pAII17中，并在预变性宿主ER2566 [pACYC-NheIM，pAII17-NheIR2]中表达。 重组克隆每克湿细胞产生约1000万单位的NheI。