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公开(公告)号：US20070207532A1
公开(公告)日：2007-09-06
申请号：US10591159
申请日：2005-03-01
申请人： Jan Barnikov , Christopher Chidley , Thomas Gronemeyer , Christian Heinis , Hughes Jaccard , Kai Johnsson , Alexandre Juillerat , Antje Keppler
发明人： Jan Barnikov , Christopher Chidley , Thomas Gronemeyer , Christian Heinis , Hughes Jaccard , Kai Johnsson , Alexandre Juillerat , Antje Keppler
IPC分类号： C12N9/00
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 06-alkylguanine substrates; (1) reduced reactivity against DNA-based substrates; and (j) reduced reactivity against N9-substituted 06-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。
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公开(公告)号：US07888090B2
公开(公告)日：2011-02-15
申请号：US10591159
申请日：2005-03-01
申请人： Jan Barnikow , Christopher Chidley , Thomas Gronemeyer , Christian Heinis , Hughes Jaccard , Kai Johnsson , Alexandre Juillerat , Antje Keppler
发明人： Jan Barnikow , Christopher Chidley , Thomas Gronemeyer , Christian Heinis , Hughes Jaccard , Kai Johnsson , Alexandre Juillerat , Antje Keppler
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 O6-alkylguanine substrates; (1) reduced reactivity against DNA-based substrates; and (j) reduced reactivity against N9-substituted O6-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 1 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O6-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N9取代的O6-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1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。
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公开(公告)号：US20110165593A1
公开(公告)日：2011-07-07
申请号：US13006087
申请日：2011-01-13
申请人： Jan Barnikow , Christopher Chidley , Thomas Gronemeyer , Christian Heinis , Hughes Jaccard , Kai Johnsson , Alexandre Juillerat , Antje Keppler
发明人： Jan Barnikow , Christopher Chidley , Thomas Gronemeyer , Christian Heinis , Hughes Jaccard , Kai Johnsson , Alexandre Juillerat , Antje Keppler
IPC分类号： G01N33/573 , 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 O6-alkylguanine substrates; (i) reduced reactivity against DNA-based substrates; and (j) reduced reactivity against N9-substituted O6-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 1 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O6-烷基鸟嘌呤底物的反应性; （i）降低对DNA基底层的反应性; 和（j）对N9取代的O6-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1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。
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公开(公告)号：US20060292651A1
公开(公告)日：2006-12-28
申请号：US10529647
申请日：2003-10-01
申请人： Alexandre Juillerat , Antje Keppler , Kai Johnsson , Thomas Gronemeier , Susanne Gendreizig , Andreas Brecht
发明人： Alexandre Juillerat , Antje Keppler , Kai Johnsson , Thomas Gronemeier , Susanne Gendreizig , Andreas Brecht
CPC分类号： C12N15/62 , C12N9/1085 , C12Q1/48
摘要： The present invention relates to methods of transferring a label from suitable substrates to O6-alkylguanine-DNA alkyltransferase (AGT) fusion proteins, to suitable fusion proteins, to suitable variants of AGT, and to novel labelled fusion proteins obtained. A protein of interest is incorporated into an AGT fusion protein, the AGT fusion protein is contacted with an AGT substrate carrying a label, and the AGT fusion protein is detected and/or manipulated using the label in a system designed for recognising and/or handling the label.
摘要翻译： 本发明涉及将标记从合适的底物转移到O6-烷基鸟嘌呤-DNA烷基转移酶（AGT）融合蛋白，合适的融合蛋白，合适的AGT变体和获得的新型标记的融合蛋白的方法。 将感兴趣的蛋白质并入AGT融合蛋白中，AGT融合蛋白与携带标记的AGT底物接触，并且使用设计用于识别和/或处理的系统中的标记来检测和/或操纵AGT融合蛋白 标签。
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公开(公告)号：US07939284B2
公开(公告)日：2011-05-10
申请号：US10474796
申请日：2002-04-05
申请人： Kai Johnsson , Susanne Gendreizig , Antje Keppler
发明人： Kai Johnsson , Susanne Gendreizig , Antje Keppler
IPC分类号： G01N33/533 , C12Q1/48 , C12P21/00 , C12P21/04 , G01N33/53
CPC分类号： G01N33/531 , C07K2319/20 , C07K2319/60
摘要： A method using O6-alkylguanine-DNA alkyltransferases (AGT) is disclosed for transferring a label from a substrate to a fusion protein comprising the AGT. This allows the detection and/or manipulating of the fusion protein, both in vitro and in vivo, by attaching molecules to the fusion proteins that introduce a new physical or chemical property to the fusion protein. Examples of such molecules are, among others, spectroscopic probes or reporter molecules, affinity tags, molecules generating reactive radicals, cross-linkers, ligands mediating protein-protein interactions or molecules suitable for the immobilisation of the fusion protein.
摘要翻译： 公开了使用O6-烷基鸟嘌呤-DNA烷基转移酶（AGT）的方法，用于将标记从底物转移至包含AGT的融合蛋白。 这允许在体外和体内通过将融合蛋白连接到融合蛋白上来检测和/或操纵融合蛋白，所述融合蛋白向融合蛋白引入新的物理或化学性质。 此类分子的实例尤其是光谱探针或报告分子，亲和标签，产生反应性基团的分子，交联剂，介导蛋白质 - 蛋白质相互作用的配体或适合固定融合蛋白的分子。
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公开(公告)号：US08367361B2
公开(公告)日：2013-02-05
申请号：US13012234
申请日：2011-01-24
CPC分类号： G01N33/531 , C07K2319/20 , C07K2319/60
摘要： A method of using O6-alkylguanine-DNA alkyltransferase (AGT) is disclosed for transferring a label from a substrate to a fusion protein comprising the AGT. This allows the detection and/or manipulating of the fusion protein, both in vitro and in vivo, by attaching molecules to the fusion proteins that introduce a new physical or chemical property to the fusion protein. Examples of such molecules are, among others, spectroscopic probes or reporter molecules, affinity tags, molecules generating reactive radicals, cross-linkers, ligands mediating protein-protein interactions or molecules suitable for the immobilisation of the fusion protein.
摘要翻译： 公开了使用O6-烷基鸟嘌呤-DNA烷基转移酶（AGT）的方法，用于将标记从底物转移至包含AGT的融合蛋白。 这允许在体外和体内通过将融合蛋白连接到融合蛋白上来检测和/或操纵融合蛋白，所述融合蛋白向融合蛋白引入新的物理或化学性质。 此类分子的实例尤其是光谱探针或报告分子，亲和标签，产生反应性基团的分子，交联剂，介导蛋白质 - 蛋白质相互作用的配体或适合固定融合蛋白的分子。
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公开(公告)号：US20110201514A1
公开(公告)日：2011-08-18
申请号：US13012234
申请日：2011-01-24
CPC分类号： G01N33/531 , C07K2319/20 , C07K2319/60
摘要： A method of using O6-alkylguanine-DNA alkyltransferase (AGT) is disclosed for transferring a label from a substrate to a fusion protein comprising the AGT. This allows the detection and/or manipulating of the fusion protein, both in vitro and in vivo, by attaching molecules to the fusion proteins that introduce a new physical or chemical property to the fusion protein. Examples of such molecules are, among others, spectroscopic probes or reporter molecules, affinity tags, molecules generating reactive radicals, cross-linkers, ligands mediating protein-protein interactions or molecules suitable for the immobilisation of the fusion protein.
摘要翻译： 公开了使用O6-烷基鸟嘌呤-DNA烷基转移酶（AGT）的方法，用于将标记从底物转移至包含AGT的融合蛋白。 这允许在体外和体内通过将融合蛋白连接到融合蛋白上来检测和/或操纵融合蛋白，所述融合蛋白向融合蛋白引入新的物理或化学性质。 此类分子的实例尤其是光谱探针或报告分子，亲和标签，产生反应性基团的分子，交联剂，介导蛋白质 - 蛋白质相互作用的配体或适合固定融合蛋白的分子。
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公开(公告)号：US20120237961A1
公开(公告)日：2012-09-20
申请号：US13484954
申请日：2012-05-31
CPC分类号： G01N33/581 , C07D239/47 , C07D403/14 , C07D405/12 , C07D487/04 , C12N9/12 , C12Q1/48
摘要： The invention relates to new proteins called alkylcytosine transferases (ACTs) derived from O6-alkylguanine-DNA alkyltransferase, and to substrates for ACTs specifically transferring a label to these ACTs and to fusion proteins comprising these. The substrates according of the invention are substituted cytosines of formula (I) wherein R1 is an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group with the double bond connected to OCH2—; R2 is a linker; and L is a label or a plurality of same or different labels. The invention further relates to methods of transferring label L from these substrates of formula (I) to ACTs and ACT fusion proteins. The system of ACT-compound of formula (I) is particularly suitable for double labelling studies together with the known system O6-alkylguanine-DNA alkyltransferase (AGT)-benzylguanines.
摘要翻译： 本发明涉及从O6-烷基鸟嘌呤-DNA烷基转移酶衍生的称为烷基胞嘧啶转移酶（ACT）的新蛋白质，以及将这些ACT特异性转移标记物的ACT和包含这些酶的融合蛋白质的底物。 根据本发明的底物是其中R 1是芳族或杂芳族基团的式（I）的取代的胞嘧啶或具有与OCH 2连接的双键的任选取代的不饱和烷基，环烷基或杂环基; R2是连接体; L是标签或多个相同或不同的标签。 本发明还涉及将标记L从这些式（I）底物转移到ACT和ACT融合蛋白的方法。 式（I）的ACT-化合物的系统特别适用于已知的系统O6-烷基鸟嘌呤-DNA烷基转移酶（AGT） - 苄基胍的双重标记研究。
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公开(公告)号：US08623627B2
公开(公告)日：2014-01-07
申请号：US13484954
申请日：2012-05-31
IPC分类号： C12N9/12 , C07D239/47
CPC分类号： G01N33/581 , C07D239/47 , C07D403/14 , C07D405/12 , C07D487/04 , C12N9/12 , C12Q1/48
摘要： The invention relates to new proteins called alkylcytosine transferases (ACTs) derived from O6-alkylguanine-DNA alkyltransferase, and to substrates for ACTs specifically transferring a label to these ACTs and to fusion proteins comprising these. The substrates according of the invention are substituted cytosines of formula (I) wherein R1 is an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group with the double bond connected to OCH2—; R2 is a linker; and L is a label or a plurality of same or different labels. The invention further relates to methods of transferring label L from these substrates of formula (I) to ACTs and ACT fusion proteins. The system of ACT-compound of formula (I) is particularly suitable for double labelling studies together with the known system O6-alkylguanine-DNA alkyltransferase (AGT)-benzylguanines.
摘要翻译： 本发明涉及从O6-烷基鸟嘌呤-DNA烷基转移酶衍生的称为烷基胞嘧啶转移酶（ACT）的新蛋白质，以及将这些ACT特异性转移标记物的ACT和包含这些酶的融合蛋白质的底物。 根据本发明的底物是其中R 1是芳族或杂芳族基团的式（I）的取代的胞嘧啶或具有与OCH 2连接的双键的任选取代的不饱和烷基，环烷基或杂环基; R2是连接体; L是标签或多个相同或不同的标签。 本发明还涉及将标记L从这些式（I）底物转移到ACT和ACT融合蛋白的方法。 式（I）的ACT-化合物的系统特别适用于已知的系统O6-烷基鸟嘌呤-DNA烷基转移酶（AGT） - 苄基胍的双重标记研究。
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公开(公告)号：US08227602B2
公开(公告)日：2012-07-24
申请号：US12309554
申请日：2007-07-24
IPC分类号： C07D239/02
CPC分类号： G01N33/581 , C07D239/47 , C07D403/14 , C07D405/12 , C07D487/04 , C12N9/12 , C12Q1/48
摘要： The invention relates to new proteins called alkylcytosine transferases (ACTs) derived from O6-alkylguanine-DNA alkyltransferase, and to substrates for ACTs specifically transferring a label to these ACTs and to fusion proteins comprising these. The substrates according of the invention are substituted cytosines of formula (I) wherein R1 is an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group with the double bond connected to OCH2—; R2 is a linker; and L is a label or a plurality of same or different labels. The invention further relates to methods of transferring label L from these substrates of formula (I) to ACTs and ACT fusion proteins. The system of ACT-compound of formula (I) is particularly suitable for double labelling studies together with the known system O6-alkylguanine-DNA alkyltransferase (AGT)-benzylguanines.
摘要翻译： 本发明涉及从O6-烷基鸟嘌呤-DNA烷基转移酶衍生的称为烷基胞嘧啶转移酶（ACT）的新蛋白质，以及特异性将标记转移至这些ACT的ACT的底物和包含它们的融合蛋白。 根据本发明的底物是其中R 1是芳族或杂芳族基团的式（I）的取代的胞嘧啶或具有与OCH 2连接的双键的任选取代的不饱和烷基，环烷基或杂环基; R2是连接体; L是标签或多个相同或不同的标签。 本发明还涉及将标记L从这些式（I）底物转移到ACT和ACT融合蛋白的方法。 式（I）的ACT-化合物的系统特别适用于已知的系统O6-烷基鸟嘌呤-DNA烷基转移酶（AGT） - 苄基胍的双重标记研究。
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