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31. 发明授权

US06589769B1 Method for cloning and expression of TspRI restriction endonuclease and TspRI methylase in E. coli 有权
标题翻译：在大肠杆菌中克隆和表达TspRI限制性内切核酸酶和TspRI甲基化酶的方法
公开(公告)号：US06589769B1
公开(公告)日：2003-07-08
申请号：US10037927
申请日：2001-10-19
申请人： Shuang-yong Xu , Andrew Dore , Michael Dalton , Jack Benner
发明人： Shuang-yong Xu , Andrew Dore , Michael Dalton , Jack Benner
IPC分类号： C12N922
CPC分类号： C12N9/22 , C12N9/1007
摘要： The present invention relates to recombinant DNA that encodes the TspRI restriction endonuclease as well as TspRI methylase, expression of TspRI restriction endonuclease and TspRI methylase in E. coli cells containing the recombinant DNA.
摘要翻译：本发明涉及编码TspRI限制性内切核酸酶以及TspRI甲基化酶的重组DNA，TspRI限制性内切核酸酶和TspRI甲基化酶在含有重组DNA的大肠杆菌细胞中的表达。

32. 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。

33. 发明授权

US06440715B1 Method for cloning and expression of Rhodothermus Obamensis DNA polymerase I large fragment in E. coli 失效
标题翻译：在大肠杆菌中克隆和表达Rhodothermus Obamensis DNA聚合酶Ⅰ大片段的方法
公开(公告)号：US06440715B1
公开(公告)日：2002-08-27
申请号：US09267311
申请日：1999-03-12
申请人： Shuang-yong Xu
发明人： Shuang-yong Xu
IPC分类号： C12N912
CPC分类号： C12N9/1252
摘要： The present invention provides a novel thermostable DNA polymerase I obtainable from Rhodothermus obamensis, which possesses 3′-5′ exonuclease activity and has a half-life of about 35 minutes at 94° C. This polymerase also contains a tyrosine residue in the ribosome binding site which improves incorporation of dideoxyribonucleic acids. Also provided are isolated DNA and vectors encoding this polymerase, as well as its large fragment, and methods for producing recombinant enzyme using the same.
摘要翻译：本发明提供了可从Rhodothermus obamensis获得的新型热稳定DNA聚合酶I，其具有3'-5'核酸外切酶活性，并且在94℃具有约35分钟的半衰期。该聚合酶还在核糖体结合中含有酪氨酸残基改善二脱氧核糖核酸结合的位点。还提供了分离的DNA和编码该聚合酶的载体，以及其大片段，以及使用其制备重组酶的方法。

34. 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））。

35. 发明授权

US06027929A Method for cloning and producing the NspI restriction endonuclease in E. coli and purification of the recombinant NspI restriction endonuclease 有权
标题翻译：在大肠杆菌中克隆和产生NspI限制性内切核酸酶的方法，并重组NspI限制性内切核酸酶的纯化
公开(公告)号：US06027929A
公开(公告)日：2000-02-22
申请号：US135782
申请日：1998-08-18
申请人： Shuang-yong Xu
发明人： Shuang-yong Xu
IPC分类号： C12N9/22 , C12N15/55
CPC分类号： C12N9/22
摘要： The present invention relates to clones which express the recombinant NspI restriction endonuclease using a NspI methylase premodified E. coli K strain RR1 (.lambda.DE3) and to methods for producing and purifying said enzyme.
摘要翻译：本发明涉及使用NspI甲基化酶预变性大肠杆菌K株RR1（λDE3）表达重组NspI限制性内切核酸酶的克隆以及用于产生和纯化所述酶的方法。

36. 发明授权

US5962296A Method for cloning and producing Thermomicrobium roseum DNA polymerase I in E. coli 失效
标题翻译：用于在大肠杆菌中克隆和产生热单孢菌DNA聚合酶I的方法
公开(公告)号：US5962296A
公开(公告)日：1999-10-05
申请号：US28361
申请日：1998-02-24
申请人： Laurence Ettwiller , Shuang-yong Xu
发明人： Laurence Ettwiller , Shuang-yong Xu
IPC分类号： C12N9/12 , C12N15/54
CPC分类号： C12N9/1252
摘要： The present invention relates to isolating DNA coding for DNA polymerase I from Thermomicrobium roseum, expressing the T. roseum DNA polymerase I gene in E. coli and purifying the recombinant T. roseum DNA polymerase I from E. coli cell extract.
摘要翻译：本发明涉及从大肠杆菌中分离出来自Thermomicrobium roseum的DNA聚合酶I分离DNA，表达T. roseum DNA聚合酶I基因，从大肠杆菌细胞提取物中纯化重组T. roseum DNA聚合酶I.

37. 发明授权

US5885818A Method for cloning and producing ageI restriction endonuclease in E. coli 失效
标题翻译：在大肠杆菌中克隆和产生ageI限制性内切核酸酶的方法
公开(公告)号：US5885818A
公开(公告)日：1999-03-23
申请号：US78459
申请日：1998-05-14
申请人： Shuang-yong Xu , Robert E. Maunus , Keith D. Lunnen , Rachel Allen
发明人： Shuang-yong Xu , Robert E. Maunus , Keith D. Lunnen , Rachel Allen
IPC分类号： C12N15/09 , C12N1/15 , C12N1/19 , C12N1/21 , C12N5/10 , C12N9/16 , C12N9/22 , C12N15/55
CPC分类号： C12N9/22
摘要： The present invention relates to recombinant DNA which encodes the AgeI restriction endonuclease as well as AgeI methyltransferase, and production of AgeI restriction endonuclease from E. coli cells containing the recombinant DNA.
摘要翻译：本发明涉及编码AgeI限制性内切核酸酶以及AgeI甲基转移酶的重组DNA，以及含有重组DNA的大肠杆菌细胞产生AgeI限制性内切核酸酶。

38. 发明授权

US5866398A Method for cloning and producing the BS1I restriction endonuclease in E. coli 失效
标题翻译：在大肠杆菌中克隆和产生Bs1I限制性内切核酸酶的方法
公开(公告)号：US5866398A
公开(公告)日：1999-02-02
申请号：US951871
申请日：1997-10-17
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： The methylase selection method was used to clone the BslI methylase gene (bslIM) from Bacillus species. A partially active BslI methylase lacking the 17 amino acid residues at the N-terminus was cloned in E. coli using expression vector pRRS. Inverse PCR was used to clone the missing portion of the BslI methylase. After cloning the complete BslI methylase gene and its upstream DNA sequences, a RadC homolog was found upstream of the BslI methylase. Because methylase gene and restriction endonuclease gene are located in proximity to each other in a particular restriction-modification system, efforts were made to clone the downstream DNA by inverse PCR. After two round of inverse PCR reactions two open reading frames (ORF) were found downstream of the BslI methylase gene. Expression of the first ORF (ORF1) in a T7 expression vector did not yield any active BslI endonuclease. Expression of the second ORF (ORF2) in E. coli and assay of the crude cell extract indicated that this gene product has DNA nicking activity. The gene product of ORF2 alone does not constitute BslI endonuclease activity. Expression of ORF1 and ORF2 in the same E. coli cell produces BslI endonuclease activity. BslI endonuclease activity can be reconstituted in vitro by mixing gene product of ORF1 and ORF2 together.
摘要翻译：甲基化酶选择方法用于从芽孢杆菌属物种克隆BslI甲基化酶基因（bslIM）。使用表达载体pRRS将在N末端缺少17个氨基酸残基的部分活性的BslI甲基化酶克隆到大肠杆菌中。逆PCR用于克隆缺失部分的BslI甲基化酶。克隆了完整的BslI甲基化酶基因及其上游DNA序列后，发现了一个RadC同源物，位于BslI甲基化酶的上游。由于甲基化酶基因和限制性内切核酸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克隆下游DNA。在两轮反向PCR反应后，发现两个开放阅读框（ORF）在BslI甲基化酶基因的下游。第一个ORF（ORF1）在T7表达载体中的表达没有产生任何活性的BslI内切核酸酶。在大肠杆菌中表达第二个ORF（ORF2），粗细胞提取物的测定表明该基因产物具有DNA切口活性。 ORF2单独的基因产物不构成BslI内切核酸酶活性。 ORF1和ORF2在同一大肠杆菌细胞中的表达产生BslI内切核酸酶活性。通过将ORF1和ORF2的基因产物混合在一起，能体外重组BslI内切核酸酶活性。

39. 发明授权

US5786195A Method for cloning and producing the bssHII restriction endonuclease in E. coli 失效
标题翻译：在大肠杆菌中克隆和产生bssHII限制性内切核酸酶的方法
公开(公告)号：US5786195A
公开(公告)日：1998-07-28
申请号：US815688
申请日：1997-03-12
申请人： Shuang-yong Xu , Jian-ping Xiao
发明人： Shuang-yong Xu , Jian-ping Xiao
IPC分类号： C12N15/09 , C12N1/21 , C12N9/10 , C12N9/16 , C12N9/22 , C12N15/00 , C12N15/54 , C12N15/55 , C12R1/07 , C12R1/19
CPC分类号： C12N9/22
摘要： The present invention relates to cloning recombinant DNA molecules encoding a multi-specific methylase gene (bssHIIM1), BssHII restriction endonuclease gene (bssHIIR), and the cognate BssHII methylase gene (bssHIIM2) from Bacillus stearothermophilus H3 E. coli. The BssHII multi-specific methylase gene was first cloned in a Sau3AI library using a modified pLITMUS28 vector (New England Biolabs, Inc., Beverly, Mass.) with two BssHII sites. Expression of the multi-specific BssHII methylase renders the two BssHII sites resistant to BssHII digestion. Surprisingly, the cloned methylase also modifies some other sites in addition to BssHII site (5'GCGCGC3'). The methylase also modifies BsrFI site (5'RCCGGY3') and HaeII site (5'RGCGCY3'); and partially modifies EagI site (5'CGGCCG3') and MIul site (5'ACGCGT3'). The beginning of the bssHIIR gene was cloned by using two degenerate primers based on the N-terminal amino acid sequence in PCR. The rest of the bssHIIR gene was cloned by inverse PCR. The cognate bssHIIM2 gene was cloned by inverse PCR and PCR. The BssHII restriction endonuclease gene was expressed in E. coli host ER417 carrying three plasmids pLysP, pLG-BssHIIM2, pET21AT-BssHIIR.
摘要翻译：本发明涉及从嗜热脂肪芽孢杆菌H3大肠杆菌克隆编码多特异性甲基化酶基因（bssHIIM1），BssHII限制性内切核酸酶基因（bssHIIR）的重组DNA分子和同源BssHII甲基化酶基因（bssHIIM2）。首先使用具有两个BssHII位点的修饰的pLITMUS28载体（New England Biolabs，Inc.，Beverly，Mass。），将BssHII多特异性甲基化酶基因克隆到Sau3AI文库中。多特异性BssHII甲基化酶的表达使两个BssHII位点抵抗BssHII消化。令人惊讶的是，除了BssHII位点（5'GCGCGC3'）之外，克隆的甲基化酶还修饰了一些其他位点。甲基化酶也修饰BsrFI位点（5'RCCGGY3'）和HaeII位点（5'RGCGCY3'）; 并部分修饰EagI位点（5'CGGCCG3'）和MIul位点（5'ACGCGT3'）。通过使用基于PCR中的N-末端氨基酸序列的两个简并引物克隆bssHIIR基因的开始。通过反向PCR克隆其余的bssHIIR基因。通过逆PCR和PCR克隆了同源bssHIIM2基因。 BssHII限制性内切核酸酶基因在携带三种质粒pLysP，pLG-BssHIIM2，pET21AT-BssHIIR的大肠杆菌宿主ER417中表达。

40. 发明授权

US5532153A Method for cloning and producing the SacI restriction endonuclease 失效
标题翻译：克隆和产生SacI限制性内切核酸酶的方法
公开(公告)号：US5532153A
公开(公告)日：1996-07-02
申请号：US409199
申请日：1995-03-23
申请人： Shuang-yong Xu , Jianping Xiao
发明人： Shuang-yong Xu , Jianping Xiao
IPC分类号： C12N15/09 , C07H21/04 , C12N1/21 , C12N9/16 , C12N9/22 , C12N15/00 , C12N15/55 , C12R1/19 , C12R1/465
CPC分类号： C12N9/22
摘要： The present invention relates to recombinant DNA which encodes the SacI restriction endonuclease and modification methylase, and production of SacI restriction endonuclease from the recombinant DNA.
摘要翻译：本发明涉及编码SacI限制性内切核酸酶和修饰甲基化酶的重组DNA，以及从重组DNA产生SacI限制性内切核酸酶。

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