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    Exonuclease-mediated nucleic acid reassembly in directed evolution
    3.
    发明授权
    Exonuclease-mediated nucleic acid reassembly in directed evolution 失效
    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。

    End selection in directed evolution
    4.
    发明授权
    End selection in directed evolution 有权

    公开(公告)号：US06713282B2

    公开(公告)日：2004-03-30

    申请号：US10099816

    申请日：2002-03-14

    申请人： Jay M. Short ,  Gerhard Johann Frey

    发明人： Jay M. Short ,  Gerhard Johann Frey

    IPC分类号： C12P2106

    CPC分类号： C07K14/445 ,  A61K39/00 ,  A61K2039/53 ,  C12N9/00 ,  C12N9/14 ,  C12N9/16 ,  C12N9/88 ,  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 end-selection-based methods is the ability to recover full-length polynucleotides from a library of progeny molecules generated by mutagenesis methods. 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.

    Exonuclease-mediated nucleic acid reassembly in directed evolution
    5.
    发明授权
    Exonuclease-mediated nucleic acid reassembly in directed evolution 有权
    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。

    End selection in directed evolution
    6.
    发明授权
    End selection in directed evolution 有权
    定向演化中的终极选择

    公开(公告)号：US06238884B1

    公开(公告)日：2001-05-29

    申请号：US09267118

    申请日：1999-03-09

    申请人： Jay M. Short ,  Gerhard Johann Frey

    发明人： Jay M. Short ,  Gerhard Johann Frey

    IPC分类号： C12P2106

    CPC分类号： C12Q1/6811 ,  C12N15/102 ,  C12N15/1058

    摘要： A directed evolution process comprising novel methods for generating improved progeny molecules having desirable properties, including, for example, a method for rapid and facilitated production from a parental polynucleotide template, of a set of mutagenized progeny polynucleotides wherein at least one codon encoding each of the 20 naturally encoded amino acids is represented at each original codon position. This method, termed site-saturation mutagenesis, or simply saturation mutagenesis, is preferably based on the use of the degenerate N,N,G/T sequence. Also, a method of producing from a parental polypeptide template, a set of mutagenized progeny polypeptides wherein each of the 20 naturally encoded amino acids is represented at each original amino acid position. Also, other mutagenization processes that can be used in combination with, or in lieu of, saturation mutagenesis, including, for example: (a) assembly and/or reassembly of polynucloetide building blocks, which building blocks can be sections of genes &/or of gene families; and (b) introduction of two or more related polynucleotides into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment. Also, vector and expression vehicles including such polynucleotides and correspondingly expressed polypeptides. Also molecular property screening methods, including a preferred method, termed end selection, comprised of using an enzyme, such as a topoisomerase, a restriction endonuclease, &/or a nicking enzyme (such as N. BstNB I), to detect a specific terminal sequence in a working polynucleotide, to produce a ligatable end thereat, and to ligate and clone the working polynucleotide.

    摘要翻译： 一种定向进化过程，其包括用于产生具有期望性质的改良后代分子的新方法，包括例如一组诱变后代多核苷酸的亲本多核苷酸模板的快速和便利生产的方法，其中至少一个编码 在每个原始密码子位置表示20个天然编码的氨基酸。 该方法，称为位点饱和诱变，或简单的饱和诱变，优选基于使用简并N，N，G / T序列。 另外，从亲本多肽模板产生一组诱变的后代多肽的方法，其中在每个原始氨基酸位置表示20个天然编码氨基酸中的每一个。 此外，可以与饱和诱变组合使用或代替饱和诱变使用的其它诱变过程，包括例如：（a）组合和/或重组多核苷酸结构单元，所述构建基团可以是基因的部分和/或 的基因家族; 和（b）将两种或更多种相关多核苷酸引入合适的宿主细胞中，使得通过重组和还原重配产生杂交多核苷酸。 而且，载体和表达载体包括这种多核苷酸和相应表达的多肽。 还包括称为末端选择的分子特性筛选方法，其包括使用酶，例如拓扑异构酶，限制性内切核酸酶和/或切酶（例如N.BstNB I），以检测特异性末端 序列，以在其中产生可连接的末端，并连接和克隆工作的多核苷酸。

    End selection in directed evolution
    7.
    发明授权
    End selection in directed evolution 有权

    公开(公告)号：US06773900B2

    公开(公告)日：2004-08-10

    申请号：US10382283

    申请日：2003-03-05

    申请人： Jay M. Short ,  Gerhard Johann Frey

    发明人： Jay M. Short ,  Gerhard Johann Frey

    IPC分类号： C12P2106

    CPC分类号： C07K14/445 ,  A61K39/00 ,  A61K2039/53 ,  C12N9/00 ,  C12N9/16 ,  C12N15/102 ,  C12N15/1034 ,  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 end-selection-based methods is the ability to recover full-length polynucleotides from a library of progeny molecules generated by mutagenesis methods. 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.

    End selection in directed evolution
    8.
    发明授权
    End selection in directed evolution 失效
    定向演化中的终极选择

    公开(公告)号：US06740506B2

    公开(公告)日：2004-05-25

    申请号：US09885551

    申请日：2001-06-19

    申请人： Jay M. Short ,  Gerhard Johann Frey

    发明人： Jay M. Short ,  Gerhard Johann Frey

    IPC分类号： C12P2106

    CPC分类号： C07K14/445 ,  A61K39/00 ,  A61K2039/53 ,  C12N9/00 ,  C12N9/16 ,  C12N15/102 ,  C12N15/1034 ,  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 end-selection-based methods is the ability to recover full-length polynucleotides from a library of progeny molecules generated by mutagenesis methods. 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。 基于终端选择的方法的特别优点是从通过诱变方法产生的后代分子的文库中恢复全长多核苷酸的能力。 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。

    End selection in directed evolution
    10.
    发明授权
    End selection in directed evolution 有权
    定向演化中的终极选择

    公开(公告)号：US06696275B2

    公开(公告)日：2004-02-24

    申请号：US09867262

    申请日：2001-05-29

    申请人： Jay M. Short ,  Gerhard Johann Frey

    发明人： Jay M. Short ,  Gerhard Johann Frey

    IPC分类号： C12P2106

    CPC分类号： C12Q1/6811 ,  C12N15/102 ,  C12N15/1058

    摘要： A directed evolution process comprising novel methods for generating improved progeny molecules having desirable properties, including, for example, a method for rapid and facilitated production from a parental polynucleotide template, of a set of mutagenized progeny polynucleotides wherein at least one codon encoding each of the 20 naturally encoded amino acids is represented at each original codon position. This method, termed site-saturation mutagenesis, or simply saturation mutagenesis, is preferably based on the use of the degenerate N,N,G/T sequence. Also, a method of producing from a parental polypeptide template, a set of mutagenized progeny polypeptides wherein each of the 20 naturally encoded amino acids is represented at each original amino acid position. Also, other mutagenization processes that can be used in combination with, or in lieu of, saturation mutagenesis, including, for example: (a) assembly and/or reassembly of polynucloetide building blocks, which building blocks can be sections of genes &/or of gene families; and (b) introduction of two or more related polynucleotides into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment. Also, vector and expression vehicles including such polynucleotides and correspondingly expressed polypeptides. Also molecular property screening methods, including a preferred method, termed end selection, comprised of using an enzyme, such as a topoisomerase, a restriction endonuclease, &/or a nicking enzyme (such as N. BstNB I), to detect a specific terminal sequence in a working polynucleotide, to produce a ligatable end thereat, and to ligate and clone the working polynucleotide.

    摘要翻译： 一种定向进化过程，其包括用于产生具有期望性质的改良后代分子的新方法，包括例如一组诱变后代多核苷酸的亲本多核苷酸模板的快速和便利生产的方法，其中至少一个编码 在每个原始密码子位置表示20个天然编码的氨基酸。 该方法，称为位点饱和诱变，或简单的饱和诱变，优选基于使用简并N，N，G / T序列。 另外，从亲本多肽模板产生一组诱变的后代多肽的方法，其中在每个原始氨基酸位置表示20个天然编码氨基酸中的每一个。 此外，可以与饱和诱变组合使用或代替饱和诱变使用的其它诱变过程，包括例如：（a）组合和/或重组多核苷酸结构单元，所述构建基团可以是基因的部分和/或 的基因家族; 和（b）将两种或更多种相关多核苷酸引入合适的宿主细胞中，使得通过重组和还原重配产生杂交多核苷酸。 而且，载体和表达载体包括这种多核苷酸和相应表达的多肽。 还包括称为末端选择的分子特性筛选方法，其包括使用酶，例如拓扑异构酶，限制性内切核酸酶和/或切酶（例如N.BstNB I），以检测特异性末端 序列，以在其中产生可连接的末端，并连接和克隆工作的多核苷酸。