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1. 发明申请

WO2005085431A3 MUTANTS OF O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE 审中-公开
公开(公告)号：WO2005085431A3
公开(公告)日：2005-09-15
申请号：PCT/EP2005/050899
申请日：2005-03-01
申请人： EPFL ECOLE POLYTECHNIQUE FEDERALE DE 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
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.

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

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。

4. 发明申请

WO2002083937A2 METHODS USING O6-ALKYLGUANINE-DNA ALKYLTRANSFERASES 审中-公开
标题翻译：使用O 6 - 甘氨酸 - DNA烷基转移酶的方法
公开(公告)号：WO2002083937A2
公开(公告)日：2002-10-24
申请号：PCT/GB2002/001636
申请日：2002-04-05
申请人： ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE , KIDDLE, Simon, John , JOHNSSON, Kai , GENDREIZIG, Susanne , KEPPLER, Antje
发明人： KIDDLE, Simon, John , JOHNSSON, Kai , GENDREIZIG, Susanne , KEPPLER, Antje
IPC分类号： C12Q1/48
CPC分类号： G01N33/531 , C07K2319/20 , C07K2319/60
摘要： A method using O 6 -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O 6 - 烷基鸟嘌呤-DNA烷基转移酶（AGT）的方法将标记从底物转移到包含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。这样的分子的实例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。

5. 发明申请

WO2004031404A1 PROTEIN LABELLING WITH O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE 审中-公开
标题翻译：蛋白标记与O6-氨基甘氨酸-DNA烷基转移酶
公开(公告)号：WO2004031404A1
公开(公告)日：2004-04-15
申请号：PCT/EP2003/010859
申请日：2003-10-01
申请人： ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE (EPFL) , JUILLERAT, Alexandre , KEPPLER, Antje , JOHNSSON, Kai , GRONEMEIER, Thomas , GENDREIZIG, Susanne , BRECHT, Andreas
发明人： JUILLERAT, Alexandre , KEPPLER, Antje , JOHNSSON, Kai , GRONEMEIER, Thomas , GENDREIZIG, Susanne , BRECHT, Andreas
IPC分类号： C12Q1/48
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。

6. 发明申请

WO2002083937A3 METHODS USING O6-ALKYLGUANINE-DNA ALKYLTRANSFERASES 审中-公开
公开(公告)号：WO2002083937A3
公开(公告)日：2002-10-24
申请号：PCT/GB2002/001636
申请日：2002-04-05
申请人： ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE , KIDDLE, Simon, John , JOHNSSON, Kai , GENDREIZIG, Susanne , KEPPLER, Antje
发明人： KIDDLE, Simon, John , JOHNSSON, Kai , GENDREIZIG, Susanne , KEPPLER, Antje
IPC分类号： G01N33/532
摘要： A method using O 6 -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.

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