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    • 4. 发明申请
    • METHOD FOR MANUFACTURING A MODIFIED PEPTIDE
    • 制备改性肽的方法
    • WO2009027495A1
    • 2009-03-05
    • PCT/EP2008/061362
    • 2008-08-29
    • PROCOMCURE BIOTECH GMBHÖNDER, KamilBAUER, Johann W.
    • ÖNDER, KamilBAUER, Johann W.
    • C12N15/10C07K14/31C07K14/47
    • C12N15/1055C12N15/102
    • The present invention relates to a method for manufacturing a modified polypeptide from a first polypeptide, said modified polypeptide exhibiting altered binding properties to a target molecule and/or having a different amino acid sequence compared to a first polypeptide comprising the steps of a) providing a first cell comprising a nucleic acid molecule encoding for a first fusion polypeptide, said first fusion polypeptide comprising at least one first polypeptide and a transcriptional activation domain, and comprising optionally a nucleic acid mole cule encoding for a second fusion polypeptide, said second fusion polypeptide comprising the target mole cule or a polypeptide domain binding the target molecule and a DNA binding domain, whereby the cell further comprises a reporter gene encoding a reporter polypeptide operably linked to an upstream transcriptional regulatory sequence comprising a DNA binding site as target for the at least one first polypeptide or optionally a DNA binding site for the DNA binding domain of the second fusion polypeptide, b) cultivating the cells of step a), c) identifying at least one cell ex pressing the reporter polypeptide, d) isolating at least one nucleic acid molecule encoding for at least one first polypeptide of the at least one cell identified in step c), e) modifying the at least one nucleic acid molecule of step d) by introducing at least one mutation thus obtaining at least one modified nucleic acid molecule encoding for at least one modified polypeptide, f) introducing the at least one modified nucleic acid molecule of step e) into at least one second cell comprising optionally a nucleic acid molecule encoding for a second fusion polypeptide, said second fusion polypeptide comprising the target molecule or a polypeptide domain binding the target molecule and a DNA binding domain, and g) repeating steps a) to f) at least twice until a nucleic acid molecule encoding for a modified polypeptide is obtained and isolated in step d) exhibiting predetermined altered binding properties to the target molecule compared to the at least one first polypeptide and/or having a different amino acid sequence compared to the first polypeptide, wherein in the repeating steps a) to d) the first polypeptide is exchanged with the modified polypeptide of step e).
    • 本发明涉及一种从第一多肽制备修饰的多肽的方法,所述修饰的多肽与第一多肽相比显示改变的与靶分子的结合特性和/或具有与第一多肽相比具有不同氨基酸序列的方法,包括以下步骤:a) 第一细胞,其包含编码第一融合多肽的核酸分子,所述第一融合多肽包含至少一个第一多肽和转录活化结构域,并且任选包含编码第二融合多肽的核酸分子,所述第二融合多肽包含 所述靶分子或多肽结构域结合所述靶分子和DNA结合结构域,其中所述细胞还包含编码报告多肽的报告基因,所述报告多肽与上游转录调节序列可操作地连接,所述上游转录调控序列包含DNA结合位点作为所述至少一个 第一多肽或任选的DNA结合蛋白 g)第二融合多肽的DNA结合结构域,b)培养步骤a)的细胞,c)鉴定至少一种按压所述报道多肽的细胞,d)分离至少一种编码至少一种的核酸分子 在步骤c)中鉴定的至少一个细胞的第一多肽,e)通过引入至少一个突变来修饰步骤d)的至少一个核酸分子,从而获得编码至少一个修饰的多肽的至少一个修饰的核酸分子 f)将至少一个步骤e)的修饰的核酸分子导入到至少一个第二细胞中,任选地包含编码第二融合多肽的核酸分子,所述第二融合多肽包含靶分子或结合靶的多肽结构域 分子和DNA结合结构域,以及g)重复步骤a)至f)至少两次,直至获得编码修饰多肽的核酸分子,并在步骤 d)与第一多肽相比,与至少一种第一多肽相比,表现出与靶分子预定的改变的结合特性,和/或与第一多肽相比具有不同的氨基酸序列,其中在重复步骤a)至d)中将第一多肽与 步骤e)的修饰多肽。