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    • 55. 发明申请
    • IN VIVO DETECTION OF PROTEINS INTERACTION BASED ON ADENYLATE CYCLASE HYBRID SYSTEM
    • 基于ADENYLATE CYCLASE HYBRID SYSTEM的蛋白质相互作用检测
    • WO2016008949A1
    • 2016-01-21
    • PCT/EP2015/066207
    • 2015-07-15
    • INSTITUT PASTEURCENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS)
    • LADANT, DanielDAVI, Marilyne
    • C12N15/10G01N33/68
    • C12N15/1055C07K14/4728C12N9/88C12Y406/01001G01N33/573G01N2333/4727G01N2333/988
    • The present invention relates to a method to detect the interaction between a target ligand and a moiety of interest using an adenylate cyclase enzyme (AC) and calmodulin (CaM) as interacting partners, said method comprising: i) expressing in a suitable host cell: (a) a low number of molecules of a first chimeric polypeptide containing AC, and (b) a low number of molecules of a second chimeric polypeptide containing CaM, wherein said AC in said first chimeric polypeptide and/or said CaM in said second chimeric polypeptide has decreased affinity for its interacting partner, wherein said AC in said first chimeric polypeptide is fused to a moiety of interest and said CaM in said second chimeric polypeptide is fused to a target ligand, or conversely, and wherein, when said moiety of interest and said target ligand interact, said AC is activated, and ii) detecting the activation of said AC. The present inventors herein show that only one AC/CaM complex per cell is sufficient to confer a selectable trait to the host cell. Unexpectedly, even less than one AC/CaM complex per cell can be sufficient to confer a selectable trait to the host cell. This surprising result confers a very high sensitivity, that is helpful for screening high affinity interactions, such as antigen-antibody interactions. Moreover, the bw expression of the chimeric proteins that is achieved in the present invention allows to characterize toxic moieties, what was not possible before.
    • 本发明涉及使用腺苷酸环化酶(AC)和钙调蛋白(CaM)作为相互作用的伴侣来检测目标配体和感兴趣部分之间的相互作用的方法,所述方法包括:i)在合适的宿主细胞中表达: (a)少量含有AC的第一嵌合多肽的分子,和(b)少量含有CaM的第二嵌合多肽的分子,其中所述第一嵌合多肽中的所述AC和/或所述第二嵌合多肽中的所述CaM 多肽对其相互作用的配偶体具有降低的亲和力,其中所述第一嵌合多肽中的所述AC与感兴趣部分融合,并且所述第二嵌合多肽中的所述CaM与靶配体融合,或相反地并且其中当所述感兴趣的部分 并且所述靶配体相互作用,所述AC被激活,和ii)检测所述AC的活化。 本发明人在此显示每个细胞仅一个AC / CaM复合物足以赋予宿主细胞可选择的性状。 意外的是,每个细胞甚至少于一个AC / CaM复合物可足以赋予宿主细胞可选择的特征。 这种惊人的结果赋予了非常高的灵敏度,这有助于筛选高亲和力相互作用,例如抗原 - 抗体相互作用。 此外,在本发明中实现的嵌合蛋白的bw表达允许表征有毒部分,以前是不可能的。