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1. 发明授权

US06939689B2 Exonuclease-mediated nucleic acid reassembly in directed evolution 失效
标题翻译：定向进化中核酸外切酶介导的核酸重组
公开(公告)号：US06939689B2
公开(公告)日：2005-09-06
申请号：US10029221
申请日：2001-12-21
申请人： Jay M. Short , Tsotne David Djavakhishvili , Gerhard Johann Frey
发明人： Jay M. Short , Tsotne David Djavakhishvili , Gerhard Johann Frey
IPC分类号： A61K39/00 , C07K14/445 , C12N9/00 , C12N9/14 , C12N9/16 , C12N9/24 , C12N9/38 , C12N15/10 , C12Q1/68 , C12P21/06 , C07H21/04 , C07K17/00
CPC分类号： C07K14/445 , A61K39/00 , A61K2039/53 , C12N9/00 , C12N9/14 , C12N9/16 , C12N15/102 , C12N15/1034 , C12Q1/6811
摘要： This invention provides methods of obtaining novel polynucleotides and encoded polypeptides by the use of non-stochastic methods of directed evolution (DirectEvolution™). A particular advantage of exonuclease-mediated reassembly methods is the ability to reassemble nucleic acid strands that would otherwise be problematic to chimerize. Exonuclease-mediated reassembly methods can be used in combination with other mutagenesis methods provided herein. These methods include non-stochastic polynucleotide site-saturation mutagenesis (Gene Site Saturation Mutagenesis™) and non-stochastic polynucleotide reassembly (GeneReassembly™). This invention provides methods of obtaining novel enzymes that have optimized physical &/or biological properties. Through use of the claimed methods, genetic vaccines, enzymes, small molecules, and other desirable molecules can be evolved towards desirable properties. For example, vaccine vectors can be obtained that exhibit increased efficacy for use as genetic vaccines. Vectors obtained by using the methods can have, for example, enhanced antigen expression, increased uptake into a cell, increased stability in a cell, ability to tailor an immune response, and the like. Furthermore, this invention provides methods of obtaining a variety of novel biologically active molecules, in the fields of antibiotics, pharmacotherapeutics, and transgenic traits.
摘要翻译：本发明提供了通过使用定向进化（DirectEvolution TM）的非随机方法获得新的多核苷酸和编码的多肽的方法。外切核酸酶介导的重组方法的一个特别优点是重新组装核酸链的能力，否则这些核酸链将成为嵌合的问题。外切核酸酶介导的重组方法可以与本文提供的其它诱变方法组合使用。这些方法包括非随机多核苷酸位点饱和诱变（Gene Site Saturation Mutagenesis TM）和非随机多核苷酸重组（GeneReassembly TM）。本发明提供获得具有优化的物理和/或生物学特性的新型酶的方法。通过使用所要求保护的方法，可以将遗传疫苗，酶，小分子和其它所需分子演变成期望的性质。例如，可以获得表现出增加用作基因疫苗的功效的疫苗载体。通过使用该方法获得的载体可具有例如增强的抗原表达，增加的细胞摄取，增加细胞的稳定性，定制免疫应答的能力等。此外，本发明提供了在抗生素，药物治疗剂和转基因性状领域中获得各种新型生物活性分子的方法。

2. 发明授权

US06361974B1 Exonuclease-mediated nucleic acid reassembly in directed evolution 有权
标题翻译：定向进化中核酸外切酶介导的核酸重组
公开(公告)号：US06361974B1
公开(公告)日：2002-03-26
申请号：US09535754
申请日：2000-03-27
申请人： Jay M. Short , Tsotne David Djavakhishvili , Gerhard Johann Frey
发明人： Jay M. Short , Tsotne David Djavakhishvili , Gerhard Johann Frey
IPC分类号： C12P2106
CPC分类号： C07K14/445 , A61K39/00 , A61K2039/53 , C12N9/00 , C12N9/16 , C12N15/102 , C12N15/1027 , C12N15/1034 , C12Q1/6811 , C12Y301/11002
摘要： This invention provides methods of obtaining novel polynucleotides and encoded polypeptides by the use of non-stochastic methods of directed evolution (DirectEvolution™). A particular advantage of exonuclease-mediated reassembly methods is the ability to reassemble nucleic acid strands that would otherwise be problematic to chimerize. Exonuclease-mediated reassembly methods can be used in combination with other mutagenesis methods provided herein. These methods include non-stochastic polynucleotide site-saturation mutagenesis (Gene Site Saturation Mutagenesis™) and non-stochastic polynucleotide reassembly (GeneReassembly™). This invention provides methods of obtaining novel enzymes that have optimized physical &/or biological properties. Through use of the claimed methods, genetic vaccines, enzymes, small molecules, and other desirable molecules can be evolved towards desirable properties. For example, vaccine vectors can be obtained that exhibit increased efficacy for use as genetic vaccines. Vectors obtained by using the methods can have, for example, enhanced antigen expression, increased uptake into a cell, increased stability in a cell, ability to tailor an immune response, and the like. Furthermore, this invention provides methods of obtaining a variety of novel biologically active molecules, in the fields of antibiotics, pharmacotherapeutics, and transgenic traits.
摘要翻译：本发明提供了通过使用定向进化（DirectEvolution TM）的非随机方法获得新的多核苷酸和编码的多肽的方法。外切核酸酶介导的重组方法的一个特别优点是重新组装核酸链的能力，否则这些核酸链将成为嵌合的问题。外切核酸酶介导的重组方法可以与本文提供的其它诱变方法组合使用。这些方法包括非随机多核苷酸位点饱和诱变（Gene Site Saturation Mutagenesis TM）和非随机多核苷酸重组（GeneReassembly TM）。本发明提供获得具有优化的物理和/或生物学特性的新型酶的方法。通过使用所要求保护的方法，可以将遗传疫苗，酶，小分子和其它所需分子演变成期望的性质。例如，可以获得表现出增加用作基因疫苗的功效的疫苗载体。通过使用该方法获得的载体可具有例如增强的抗原表达，增加的细胞摄取，增加细胞的稳定性，定制免疫应答的能力等。此外，本发明提供了在抗生素，药物治疗剂和转基因性状领域中获得各种新型生物活性分子的方法。

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