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    • 99. 发明授权
    • Saturation mutageneis in directed evolution
    • 定向进化中的饱和诱变
    • US06764835B2
    • 2004-07-20
    • US10309587
    • 2002-12-04
    • Jay M. Short
    • Jay M. Short
    • C12P2106
    • C07K14/445A61K39/00A61K2039/53C12N9/00C12N9/16C12N15/102C12N15/1027C12N15/1034C12N15/1058
    • Disclosed is a rapid and facilitated method of producing from a parentlal template polynucleotide, a set of mutagenized progeny polynculeotides whereby at each original codon position there is produced at least one substitute codon encoding each of the 20 naturally encoded amino acids. Accordingly, there is also provided a method of producing from a parental template polypeptide, a set of mutagenized progeny polypeptides wherein each of the 20 naturally encoded amino acids is represented at each original amino acid position. The method provided is termed site-saturation mutagenesis, or simply saturation mutagenesis, and can be used in combination with other mutagenization processes, such as, for example, a process wherein two or more related polynucleotides are introduced into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment. Also provided are vector and expression vehicles incuding such polynucleotides, polypeptides expressed by the hybrid polynucleotides and a method for screening for hybrid polypeptides.
    • 公开了一种由亲本模板多核苷酸,一组诱变的后代多核苷酸产生的快速和便利的方法,其中在每个原始密码子位置产生编码20个天然编码氨基酸中的每一个的至少一个替代密码子。 因此,还提供了从亲本模板多肽,一组诱变的后代多肽生产其中每个原始氨基酸位置上表示20个天然编码氨基酸的方法。 所提供的方法称为位点饱和诱变或简单的饱和诱变,并且可以与其它诱变过程组合使用,例如其中将两个或更多个相关多核苷酸引入合适的宿主细胞中的方法,使得 通过重组和还原重配产生杂交多核苷酸。 还提供了包含这种多核苷酸的载体和表达载体,由杂交多核苷酸表达的多肽和杂交多肽的筛选方法。
    • 100. 发明授权
    • End selection in directed evolution
    • 定向演化中的终极选择
    • US06696275B2
    • 2004-02-24
    • US09867262
    • 2001-05-29
    • Jay M. ShortGerhard Johann Frey
    • Jay M. ShortGerhard Johann Frey
    • C12P2106
    • C12Q1/6811C12N15/102C12N15/1058
    • 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.
    • 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。