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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：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：US08163479B2
公开(公告)日：2012-04-24
申请号：US10591162
申请日：2005-03-01
IPC分类号： C07D473/18 , C07D519/00 , C07D495/04 , C07D473/40
CPC分类号： C07D473/18
摘要： The invention relates to substrates for O6-alkylguanine-DNA alkyltransferases (AGT) of formula R1-A-X—CH2—R3—R4-L1, wherein A is a group recognized by AGT as a substrate, X is oxygen or sulfur, R1 is a group —R2-L2 or a group R5, R2 and R4 are, independently of each other, a linker, R3 is an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group with the double bond connected to CH2, R5 is arylmethyl or heteroarylmethyl or an optionally substituted cycloalkyl, cycloalkenyl or heterocyclyl group, L1 is a label, a plurality of same or different labels, a bond connecting R4 to A forming a cyclic substrate, or a further group —R3—CH2—X-A-R1, and L2 is a label or a plurality of same or different labels. The invention further relates to methods of transferring a label from these substrates to O6-alkylguanine-DNA alkyltransferases (AGT) and AGT fusion proteins.
摘要翻译： 本发明涉及式R1-AX-CH2-R3-R4-L1的O6-烷基鸟嘌呤-DNA烷基转移酶（AGT）的底物，其中A为被AGT识别为底物的基团，X为氧或硫，R1为 基团-R2-L2或基团R5，R2和R4彼此独立地是连接基，R3是芳族或杂芳族基团，或任选取代的不饱和烷基，环烷基或杂环基，其双键与CH2连接 R 5是芳基甲基或杂芳基甲基或任选取代的环烷基，环烯基或杂环基，L1是标记，多个相同或不同的标记，连接R4与A形成环状底物的键或另外的基团-R 3 -CH 2 - XA-R1和L2是标签或多个相同或不同的标签。 本发明还涉及将标记从这些底物转移到O6-烷基鸟嘌呤-DNA烷基转移酶（AGT）和AGT融合蛋白的方法。
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公开(公告)号：US20070243568A1
公开(公告)日：2007-10-18
申请号：US10591162
申请日：2005-03-01
IPC分类号： C07D473/18 , C12Q1/48
CPC分类号： C07D473/18
摘要： The invention relates to substrates for O6-alkylguanine-DNA alkyltransferases (AGT) of formula R1-A-X—CH2—R3—R4-L1, wherein A is a group recognized by AGT as a substrate, X is oxygen or sulfur, R1 is a group —R2-L2 or a group R5, R2 and R4 are, independently of each other, a linker, R3 is an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group with the double bond connected to CH2, R5 is arylmethyl or heteroarylmethyl or an optionally substituted cycloalkyl, cycloalkenyl or heterocyclyl group, L1 is a label, a plurality of same or different labels, a bond connecting R4 to A forming a cyclic substrate, or a further group —R3 CH2—X-A-R1, and L2 is a label or a plurality of same or different labels. The invention further relates to methods of transferring a label from these substrates to O6-alkylguanine-DNA alkyltransferases (AGT) and AGT fusion proteins.
摘要翻译： 本发明涉及式R 1 -AX-CH 2 -R Sub的O 6 - 烷基鸟嘌呤-DNA烷基转移酶（AGT）的底物 其中A是被AGT识别为底物的基团，X是氧或硫，R 1 是基团-R 2→L 2或基团R 5，R 2和/或 R 4彼此独立地是连接基，R 3是芳族或杂芳族基团，或任选取代的不饱和烷基，环烷基或杂环基与双 与CH 2 R 2连接的键是R 5是芳基甲基或杂芳基甲基或任选取代的环烷基，环烯基或杂环基，L 1是标记， 多个相同或不同的标记，连接R 4与形成环状基底的A的键，或另外的基团-R 3 3 CH 2 -XAR 1＆lt; 1＆gt;和L 2 2是标签或多个相同或不同的标签。 本发明还涉及将标记从这些底物转移到-O6-6-烷基鸟嘌呤-DNA烷基转移酶（AGT）和AGT融合蛋白的方法。
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