摘要
基于生物素酶的蛋白质邻近标记技术是利用融合在感兴趣蛋白上的生物素连接酶对邻近的蛋白质生物素化,通过生物素与链酶亲和素之间的亲和力进行分离,再结合质谱分析鉴定出生物素化蛋白。该技术能够用于检测弱而短暂的蛋白质相互作用,也给在无膜细胞器和其他不易分离或纯化的亚细胞结构的上的蛋白互作研究提供了新的选择,很好地补充了传统研究蛋白质互作方法的空白。本文对近几年出现的基于生物素酶的蛋白质邻近标记的技术发展及其应用进行了综述。
蛋白质是生命体中真正发挥作用的物质,大多数蛋白质不能单独发挥作用,它们与其他蛋白质的相互作用决定了细胞的功能。因此,详细了解蛋白质间相互作用(PPI)是破译细胞网络和调控途径的关键。在用传统的亲和纯化方法分离纯化蛋白复合物时,会导致许多蛋白质间的相互作用被破坏。同时,传统方法会破坏细胞器的完整性,很难评估所得的纯化产物是否保持了其在体内的原始结
邻近标记技术是通过在需要研究的“诱饵”蛋白上融合一个能够非特异性标记周围蛋白质的酶,被打上标记的蛋白质可以形象的称为“猎物”蛋白。目前基于邻近标记酶的蛋白质标记技术有了快速的发展,有依赖生物素连接酶(BirA)建立的生物素连接酶突变体的非序列依赖标记技术(proximity-dependent biotin identification,BioID),在存在生物素的条件下BirA可以将邻近蛋白质的赖氨酸残基上带上生物素标签;依赖工程化抗坏血酸过氧化物酶(APEX)建立的生物素标记,APEX在过氧化氢的存在下可在短时间内实现快速的生物素化,有利于研究空间和时间上的蛋白质相互作用网络。依赖于PUP连接酶PafA的邻近标记技术PUP-I
BioID是一种在真核细胞中近距离依赖标记的蛋白质的技术。该方法可以将物理空间上靠近诱饵蛋白附近的蛋白标记上生物素,以作为蛋白质相互作用的证据,生物素化蛋白可通过链酶亲和素俘获分离并通过质谱鉴定。该技术的核心功能组件是生物素连接酶,在大肠杆菌中,生物素连接酶BirA(231 AA)识别的底物具有特异性,仅能生物素化一段特定的氨基酸序列。如果将BirA的118位进行突变(R118G,表示为BirA*,也被称作BioID
Split-BioID是一种基于BirA*酶的蛋白质片段互补分析方法,可以从空间和时间上分析蛋白质复合
Contact-ID也是split-BioID的一种,将BirA*分为N-G78(B1)和G79-C(B2)两个片段,并将这两个片段分别与位于ER膜(ERM)的SEC61B氮端(B1-SEC61B)和位于线粒体膜的外膜(OMM)的TOM20碳端(TOM20-B2)融
BioID2(231 AA)是来源于A.aeolicus的生物素连接酶R40G突变体,自然缺乏DNA结合结构域的生物素连接酶,比BioID小约三分之一,体积的减小增强了与其融合的蛋白的靶向性和定位
所有的标记蛋白与靶蛋白融合都需要考虑一个问题,即融合蛋白是否会影响靶蛋白的功能与定位。BioID1的大小为35 kD,有研究表明BioID1的存在可能会限制内核膜(INM)蛋白通过核孔复合物(NPC),从而干扰它们的正确靶向,在INM蛋白的核质结构域延伸超过60 ~ 70 kD时,它们进入细胞核的过程就会受到阻
为了解决这个问题,Chojnowski
TurboID和miniTurbo是由Branon
Split-TurboI
May
AirID在2020年被首次报道,是Kido
亚细胞结构的蛋白质组和转录组传统上是通过免疫共沉淀(Co-immunoprecipitation,Co-IP)和生化分离分析的。然而,这两种方法都需要先裂解细胞,这容易失去低亲和与短暂的蛋白质相互作用,同时co-IP也受到可用抗体的限制,生化分离往往做不到纯化完
病毒进入宿主细胞后,病毒-宿主蛋白相互作用是病毒生命周期进行调控的主要方
Rudolph
RAS是一个药物难以干预的癌基因,Kovalsk
邻近依赖的生物素化方法,除BioID外还有APEX,邻近生物素化标记克服了经典方法纯化的局限性,近年来在各个方面得到了广泛的应用。将过氧化物酶用于生物素化,可以在1 min内完成邻近蛋白质的标
近年来,基于生物素酶的蛋白质邻近标记技术逐渐被研究人员应用在各个研究领域。邻近标记酶不仅可以单独运用,还可以与其他方法结合,如将BioID与AP-MS融合产生的MAC-ta
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