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1. 发明申请

WO2005085431A2 MUTANTS OF O<6>-ALKYLGUANINE-DNA ALKYLTRANSFERASE 审中-公开
标题翻译： O 6 - 烷基鸟嘌呤-DNA烷基转移酶的突变体
公开(公告)号：WO2005085431A2
公开(公告)日：2005-09-15
申请号：PCT/EP2005050899
申请日：2005-03-01
申请人： EPFL ECOLE POLYTECHNIQUE FEDERALE LAUSANNE , BARNIKOV JAN , CHIDLEY CHRISTOPHER , GRONEMEYER THOMAS , HEINIS CHRISTIAN , JACCARD HUGHES , JOHNSSON KAI , JUILLERAT ALEXANDRE , KEPPLER ANTJE
发明人： BARNIKOV JAN , CHIDLEY CHRISTOPHER , GRONEMEYER THOMAS , HEINIS CHRISTIAN , JACCARD HUGHES , JOHNSSON KAI , JUILLERAT ALEXANDRE , KEPPLER ANTJE
IPC分类号： C12N9/10 , C12N15/62 , G01N33/535
CPC分类号： C12N9/10 , C07K2319/00 , C12N9/1007
摘要： The invention relates to AGT mutants showing, when compared to the wild type human AGT, two or more advantageous properties selected from (a) reduced DNA interaction; (b) localisation of the expressed protein in eukaryotic cells that is no longer restricted to the nucleus; (c) improved expression yield as soluble protein and improved stability in various hosts; (d) improved stability under oxidising conditions; (e) improved stability within cells after reaction with a substrate; (f) improved stability outside cells before and after reaction with a substrate; (g) improved in vitro solubility; (h) improved reactivity against 0 -alkylguanine substrates; (1) reduced reactivity against DNA-based substrates; and (j) reduced reactivity against N -substituted 0 -alkylguanine substrates. Such AGT mutants with the mentioned improved properties are mutants wherein between 1 and 25 amino acids of the wild type human AGT are substituted by other amino acids, and optionally I to 5 amino acids out of the continuous chain at one, two or three positions are deleted or added and/or 1 to 4 amino acids at the N-terminus or 1 to 40 amino acids at the C-terminus are deleted. The invention further relates to a method for detecting and/or manipulating a protein of interest wherein the protein of interest is incorporated into a fusion protein with the AGT mutants of the invention. Another object of the invention are AGT fusion proteins comprising such AGT mutants and the protein of interest.
摘要翻译：本发明涉及AGT突变体，当与野生型人AGT比较时，显示选自以下的两种或更多种有利特性：（a）减少的DNA相互作用; （b）表达蛋白在不再限于细胞核的真核细胞中定位; （c）作为可溶性蛋白的提高的表达产量和改善的各种宿主的稳定性; （d）在氧化条件下改善稳定性; （e）与基质反应后细胞内稳定性提高; （f）在与基质反应之前和之后改善细胞外的稳定性; （g）体外溶解度提高; （h）提高了对0-6-烷基鸟嘌呤底物的反应性; （1）对DNA基底物的反应性降低; 和（j）对N 9 - 取代的0-6-烷基鸟嘌呤底物的反应性降低。具有提到的改进性质的这种AGT突变体是这样的突变体，其中野生型人AGT的1至25个氨基酸被其他氨基酸取代，并且任选地在连续链之外的1至5个氨基酸的一个，两个或三个位置是缺失或添加，和/或N端的1至4个氨基酸或C端的1至40个氨基酸缺失。本发明进一步涉及用于检测和/或操纵感兴趣的蛋白质的方法，其中感兴趣的蛋白质与本发明的AGT突变体并入融合蛋白质中。本发明的另一个目的是包含这种AGT突变体和感兴趣的蛋白质的AGT融合蛋白。

2. 发明申请

WO2005085431A9 MUTANTS OF O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE 审中-公开
标题翻译： O6-ALKYLGUANINE-DNA烷基转移酶的突变体
公开(公告)号：WO2005085431A9
公开(公告)日：2006-10-12
申请号：PCT/EP2005050899
申请日：2005-03-01
申请人： EPFL ECOLE POLYTECHNIQUE FEDERALE LAUSANNE , BARNIKOV JAN , CHIDLEY CHRISTOPHER , GRONEMEYER THOMAS , HEINIS CHRISTIAN , JACCARD HUGHES , JOHNSSON KAI , JUILLERAT ALEXANDRE , KEPPLER ANTJE
发明人： BARNIKOV JAN , CHIDLEY CHRISTOPHER , GRONEMEYER THOMAS , HEINIS CHRISTIAN , JACCARD HUGHES , JOHNSSON KAI , JUILLERAT ALEXANDRE , KEPPLER ANTJE
IPC分类号： C12N9/10
CPC分类号： C12N9/10 , C07K2319/00 , C12N9/1007
摘要： The invention relates to AGT mutants showing, when compared to the wild type human AGT, two or more advantageous properties selected from (a) reduced DNA interaction; (b) localisation of the expressed protein in eukaryotic cells that is no longer restricted to the nucleus; (c) improved expression yield as soluble protein and improved stability in various hosts; (d) improved stability under oxidising conditions; (e) improved stability within cells after reaction with a substrate; (f) improved stability outside cells before and after reaction with a substrate; (g) improved in vitro solubility; (h) improved reactivity against 0 6 -alkylguanine substrates; (1) reduced reactivity against DNA-based substrates; and (j) reduced reactivity against N 9 -substituted 0 6 -alkylguanine substrates. Such AGT mutants with the mentioned improved properties are mutants wherein between 1 and 25 amino acids of the wild type human AGT are substituted by other amino acids, and optionally I to 5 amino acids out of the continuous chain at one, two or three positions are deleted or added and/or 1 to 4 amino acids at the N-terminus or 1 to 40 amino acids at the C-terminus are deleted. The invention further relates to a method for detecting and/or manipulating a protein of interest wherein the protein of interest is incorporated into a fusion protein with the AGT mutants of the invention. Another object of the invention are AGT fusion proteins comprising such AGT mutants and the protein of interest.
摘要翻译：本发明涉及AGT突变体，当与野生型人AGT相比时，显示出选自（a）减少的DNA相互作用的两种或更多种有利性质; （b）表达的蛋白质在不再限于细胞核的真核细胞中的定位; （c）提高作为可溶性蛋白质的表达产率和改善各种宿主的稳定性; （d）改善氧化条件下的稳定性; （e）在与底物反应后改善细胞内的稳定性; （f）在与底物反应之前和之后提高细胞外的稳定性; （g）提高体外溶解度; （h）改善对0-6个 - 烷基鸟嘌呤底物的反应性; （1）降低对DNA基底层的反应性; 和（j）对N 9 - 取代的0-6-烷基鸟嘌呤底物的反应性降低。具有上述改进性质的这种AGT突变体是突变体，其中野生型人AGT的1至25个氨基酸被其它氨基酸取代，并且在一个，两个或三个位置上，连续链中任选地I至5个氨基酸是在N-末端缺失或添加和/或1至4个氨基酸或C末端的1至40个氨基酸被缺失。本发明还涉及用于检测和/或操纵感兴趣的蛋白质的方法，其中将目的蛋白质与本发明的AGT突变体掺入融合蛋白中。本发明的另一个目的是包含这种AGT突变体和目的蛋白质的AGT融合蛋白。

3. 发明申请

WO2012058458A3 METHOD FOR INCREASING THE EFFICIENCY OF DOUBLE-STRAND BREAK-INDUCED MUTAGENESIS 审中-公开
标题翻译：增加双链断裂诱导突变效率的方法
公开(公告)号：WO2012058458A3
公开(公告)日：2013-01-10
申请号：PCT/US2011058133
申请日：2011-10-27
申请人： CELLECTIS SA , DUCHATEAU PHILIPPE , JUILLERAT ALEXANDRE , SILVA GEORGE H , EPINAT JEAN-CHARLES
发明人： DUCHATEAU PHILIPPE , JUILLERAT ALEXANDRE , SILVA GEORGE H , EPINAT JEAN-CHARLES
IPC分类号： C12N9/22 , C12N15/10
CPC分类号： C12N15/01 , C12N9/16 , C12N9/22 , C12N15/102
摘要： The present invention relates to a method for increasing double-strand break-induced mutagenesis at a genomic locus of interest in a cell, thereby giving new tools for genome engineering, including therapeutic applications and cell line engineering. More specifically, the present invention concerns a method for increasing double-strand break-induced mutagenesis at a genomic locus of interest, leading to a loss of genetic information and preventing any scarless re-ligation of said genomic locus of interest by NHEJ. The present invention also relates to engineered endonucleases, chimeric or not, vectors, compositions and kits used to implement this method.
摘要翻译：本发明涉及在细胞中感兴趣的基因座上增加双链断裂诱变的方法，从而为基因组工程提供新的工具，包括治疗应用和细胞系工程。更具体地说，本发明涉及在感兴趣的基因组轨迹上增加双链断裂诱导诱变的方法，导致遗传信息的丧失，并且防止NHEJ对所述感兴趣的所述基因组座位的任何无疤痕重新连接。本发明还涉及用于实施该方法的工程内切核酸酶，嵌合体或非嵌合体载体，组合物和试剂盒。

4. 发明申请

WO2013182910A3 TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR (TALE) FUSION PROTEIN 审中-公开
标题翻译：转录激活子效应（TALE）融合蛋白
公开(公告)号：WO2013182910A3
公开(公告)日：2014-02-13
申请号：PCT/IB2013001741
申请日：2013-06-05
申请人： CELLECTIS , JUILLERAT ALEXANDRE , DUCHATEAU PHILIPPE
发明人： JUILLERAT ALEXANDRE , DUCHATEAU PHILIPPE
IPC分类号： C12N9/22 , C12N15/10
CPC分类号： C12N15/907 , C07K2319/80 , C12N9/22 , C12N15/10
摘要： The present invention relates to Transcription Activator-Like Effector (TALE) derived proteins that allow to efficiently target and/or process double stranded nucleic acid sequences. The proteins of the invention are typically chimeric protein monomers composed of a core scaffold comprising Repeat Variable Dipeptide regions (RVDs) having binding specificity to a DNA target sequence, to which is fused a catalytic domain to its N-terminal. This later catalytic domain, which can be a monomer of a nuclease, is placed at this position to possibly interact with another catalytic domain fused to another TAL monomer, such that, when said monomers are binding to their respective target DNA sequences, both catalytic domains form a catalytic entity likely to process DNA in the proximity of these target sequences. This new TAL architecture makes it possible to target only one DNA strand, which is not the case, for instance, with classical TALEN architectures. The present invention also relates to vectors encoding such proteins and compositions or kits in which Transcription Activator-Like Effector (TALE) proteins of the present invention are used.
摘要翻译：本发明涉及允许有效靶向和/或加工双链核酸序列的转录激活子样效应子（TALE）衍生蛋白。本发明的蛋白质通常是由包含对DNA靶序列具有结合特异性的重复可变二肽区域（RVD）的核心支架构成的嵌合蛋白质单体，将催化结构域融合到其N-末端。该后续的催化结构域（其可以是核酸酶的单体）置于该位置以可能与另一个与另一个TAL单体融合的另一催化结构域相互作用，使得当所述单体与其各自的靶DNA序列结合时，两个催化结构域形成可能在这些靶序列附近处理DNA的催化实体。这种新的TAL架构使得可以仅靶向一条DNA链，而不是例如经典的TALEN架构。本发明还涉及编码这样的蛋白质的载体，其中使用本发明的转录激活剂样效应子（TALE）蛋白质的组合物或试剂盒。

5. 发明申请

WO2012138927A3 METHOD FOR THE GENERATION OF COMPACT TALE-NUCLEASES AND USES THEREOF 审中-公开
标题翻译：用于生成紧凑型陶瓷核的方法及其用途
公开(公告)号：WO2012138927A3
公开(公告)日：2013-04-25
申请号：PCT/US2012032426
申请日：2012-04-05
申请人： DUCHATEAU PHILIPPE , JUILLERAT ALEXANDRE , VALTON JULIEN , BERTONATI CLAUDIA , EPINAT JEAN-CHARLES , SILVA GEORGE H
发明人： DUCHATEAU PHILIPPE , JUILLERAT ALEXANDRE , VALTON JULIEN , BERTONATI CLAUDIA , EPINAT JEAN-CHARLES , SILVA GEORGE H
IPC分类号： A61K38/16 , C12N9/22 , C12N15/10
CPC分类号： C12N9/22 , A61K38/00 , C07K14/4703 , C07K2319/00 , C07K2319/80 , C12N9/16 , C12P19/34
摘要： The present invention relates to a method for the generation of compact Transcription Activator-Like Effector Nucleases (TALENs) that can efficiently target and process double-stranded DNA. More specifically, the present invention concerns a method for the creation of TALENs that consist of a single TALE DNA binding domain fused to at least one catalytic domain such that the active entity is composed of a single polypeptide chain for simple and efficient vectorization and does not require dimerization to target a specific single double-stranded DNA target sequence of interest and process DNA nearby said DNA target sequence. The present invention also relates to compact TALENs, vectors, compositions and kits used to implement the method.
摘要翻译：本发明涉及能够有效靶向和加工双链DNA的致密转录激活子样效应核酸酶（TALEN）的生成方法。更具体地，本发明涉及一种用于产生TALEN的方法，其由与至少一个催化结构域融合的单个TALE DNA结合结构域组成，使得活性实体由单个多肽链组成，用于简单且有效的载体化，并且不需要二聚化来靶向特定的单个双链DNA靶序列，并在DNA靶序列附近处理DNA。本发明还涉及用于实施该方法的紧凑型TALEN，载体，组合物和试剂盒。

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