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公开(公告)号：US20100075394A1
公开(公告)日：2010-03-25
申请号：US12594256
申请日：2008-04-04
CPC分类号： C07K19/00
摘要： The invention provides a method of crosslinking two objects of interest, comprising the steps of: i) providing a fusion protein comprising at least a first protein and a second protein, wherein both the first and the second protein are, based on their structure and function, capable of forming a covalent bond with given substrates, and which first and second proteins are of substantially non-overlapping substrate selectivity, preferably of different substrate specificity; ii) providing a first object of interest, comprising a substrate moiety for the first protein of the said fusion protein, and providing a second object of interest, comprising a substrate moiety for the second protein of the said fusion protein; and iii) reacting said first protein of the fusion protein with the substrate moiety of said first object, and reacting said second protein of the fusion protein with the substrate moiety of said second object, thereby covalently crosslinking the first object to the second object via the said fusion protein. Most prominent applications of the disclosed method are, due to the straight-forward, reliable, directional and fast crosslinking reactions: the derivatization of cells, antibodies and the crosslinking of proteins.
摘要翻译： 本发明提供了交联两个感兴趣对象的方法，其包括以下步骤：i）提供包含至少第一蛋白质和第二蛋白质的融合蛋白质，其中第一和第二蛋白质均基于其结构和功能 能够与给定的底物形成共价键，并且哪些第一和第二蛋白质具有基本上不重叠的底物选择性，优选不同的底物特异性; ii）提供第一感兴趣对象，其包括用于所述融合蛋白的第一蛋白质的底物部分，并提供第二感兴趣对象，其包含所述融合蛋白质的第二蛋白质的底物部分; 和iii）使所述融合蛋白的第一蛋白质与所述第一物体的底物部分反应，并使所述融合蛋白的所述第二蛋白质与所述第二物体的底物部分反应，由此通过所述第二物体共价交联所述第一物体与所述第二物体 所述融合蛋白。 所公开方法的最突出的应用是由于直接，可靠，定向和快速的交联反应：细胞的衍生，抗体和蛋白质的交联。
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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：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：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：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：US07666612B2
公开(公告)日：2010-02-23
申请号：US10557897
申请日：2004-05-19
申请人： Kai Johnsson , Nathalie George
发明人： Kai Johnsson , Nathalie George
CPC分类号： C07K14/245 , C07K2319/21 , C07K2319/43 , G01N33/535 , G01N33/581
摘要： A method for labeling acyl carrier protein (ACP) fusion proteins with a wide variety of different labels is disclosed. The method relies on the transfer of a label from a coenzyme A type substrate to an ACP fusion protein using a holo-acyl carrier protein synthase (ACPS) or a homologue thereof. The method allows detecting and manipulating 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 labels 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 immobilization of the fusion protein.
摘要翻译： 公开了用多种不同标记物标记酰基载体蛋白（ACP）融合蛋白的方法。 该方法依赖于使用全酰基载体蛋白合成酶（ACPS）或其同源物将标记从辅酶A型底物转移至ACP融合蛋白。 该方法允许在体外和体内检测和操纵融合蛋白，通过将融合蛋白连接到融合蛋白上，向融合蛋白引入新的物理或化学性质。 此类标记的实例包括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用于固定融合蛋白的分子。
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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：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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