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1. 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））。

2. 发明授权

US6066487A Method for cloning and expression of BsrFI restriction endonuclease in E. coli 有权
标题翻译：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。

3. 发明授权

US5663067A Method for cloning and producing the SapI restriction endonuclease in E. coli 失效
标题翻译：在大肠杆菌中克隆和产生SapI限制性内切核酸酶的方法
公开(公告)号：US5663067A
公开(公告)日：1997-09-02
申请号：US678304
申请日：1996-07-11
申请人： Shuang-yong Xu , Jian-ping Xiao , Robert E. Maunus
发明人： Shuang-yong Xu , Jian-ping Xiao , Robert E. Maunus
IPC分类号： C12N15/09 , C07H21/04 , C12N1/21 , C12N9/10 , C12N9/16 , C12N9/22 , C12N15/00 , C12N15/55 , C12R1/19 , C12R1/21 , C12N15/70
CPC分类号： C12N9/22
摘要： The present invention relates to recombinant DNA which encodes the SapI restriction endonuclease and modification methylase, and the production of SapI restriction endonuclease from the recombinant DNA as well as to methods for cloning Actinomycetes genes into suitable hosts such as E. coli.
摘要翻译：本发明涉及编码SapI限制性内切核酸酶和修饰甲基化酶的重组DNA，以及来自重组DNA的SapI限制性内切核酸酶的产生以及将放线菌基因克隆到合适宿主如大肠杆菌中的方法。

4. 发明授权

US06387681B1 Method for cloning and expression of NHEI restriction endonuclease in E. coli. 有权
标题翻译：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。

5. 发明授权

US6130078A Method for cloning the NSPHI restriction-modification system in E. coli and producing the recombinant NSPHI restriction endonuclease 有权
标题翻译：在大肠杆菌中克隆NSPHI限制性修饰系统并产生重组NSPHI限制性内切核酸酶的方法
公开(公告)号：US6130078A
公开(公告)日：2000-10-10
申请号：US193191
申请日：1998-11-17
申请人： Jian-ping Xiao , Shuang-yong Xu
发明人： Jian-ping Xiao , Shuang-yong Xu
IPC分类号： C12N9/22 , C12N15/55
CPC分类号： C12N9/22
摘要： The present invention relates to recombinant DNA molecules encoding NspHI restriction endonuclease and methylase and to method to use premodified E. coli K strain RR1 (.lambda.DE3) for overexpression of NspHI restriction endonuclease.
摘要翻译：本发明涉及编码NspHI限制性内切核酸酶和甲基化酶的重组DNA分子以及使用预变性大肠杆菌K株RR1（λDE3）用于NspHI限制性内切核酸酶过表达的方法。

6. 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。

7. 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。

8. 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。

9. 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。

10. 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。

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