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1. 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。

2. 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。

3. 发明申请

WO2005085470A9 SPECIFIC SUBSTRATES FOR O6- ALKYLGUANINE-DNA ALKYLTRANSFERASE 审中-公开
标题翻译：用于O6-氨基葡萄糖-DNA烷基转移酶的特异性底物
公开(公告)号：WO2005085470A9
公开(公告)日：2006-10-05
申请号：PCT/EP2005050900
申请日：2005-03-01
申请人： EPFL ECOLE POLYTECHNIQUE FEDERALE LAUSANNE , JACCARD HUGHES , JOHNSSON KAI , KINDERMANN MAIK , SIELAFF INDIA CHRISTINA
发明人： JACCARD HUGHES , JOHNSSON KAI , KINDERMANN MAIK , SIELAFF INDIA CHRISTINA
IPC分类号： C07D473/18 , C12Q1/48
CPC分类号： C07D473/18
摘要： The invention relates to substrates for O 6 -alkylguanine-DNA alkyltransferases (AGT) of formula R 1 -A-X-CH 2 -R 3 -R 4 -L 1 , wherein A is a group recognized by AGT as a substrate, X is oxygen or sulfur, R 1 is a group -R 2 -L 2 or a group R 5 , R 2 and R 4 are, independently of each other, a linker, R 3 is an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group with the double bond connected to CH 2 , R 5 is arylmethyl or heteroarylmethyl or an optionally substituted cycloalkyl, cycloalkenyl or heterocyclyl group, L 1 is a label, a plurality of same or different labels, a bond connecting R 4 to A forming a cyclic substrate , or a further group -R 3 CH 2 -X-A-R 1, and L 2 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 O 6-
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; / 2＆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的方法

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