可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体(SNAER)家族蛋白是大多数细胞内和胞吐膜融合途径的引擎(Jahn和Scheller,2006)。在过去的二十年里,体外脂质体融合已被证明是重建生理SNARE介导的膜融合过程的强大工具(Liuetal.2017)。膜融合过程的重建不仅为同源SNARE复合物驱动膜融合的能力提供了直接证据,而且还使研究人员能够研究相关途径中调节蛋白的功能机制(Wickner和Rizo2017)。在此之前,已经建立了多种用于体外SNARE介导的脂质体融合的精细方法(Bao等人。2018年;Diao等人。2012;Duzgunes2003;Gong等人。2015年;Heo等人。2021年;Kiessling等人。2015年;Kreye等人。2008;Kyoung等人。2013;Liu等人。2017年;斯科特等人。2003).尽管技术进步使重建在生理上更具相关性,越来越复杂的实验程序,仪器,和数据处理算法仍然阻碍了非专家建立基本的SNARE介导的脂质体融合测定法。这里,我们描述了一个低成本的,节省时间,和易于处理的方案,以根据我们以前的出版物建立基础的体外SNARE介导的脂质体融合测定(Liu等人。2023年;王和马2022年)。该方案可以通过使用适当的替代添加剂(例如,蛋白质,大分子,化学品,等。).一轮测定所需的总时间通常为两天,并且可以极度压缩为一天。
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNAER) family proteins are the engines of most intra-cellular and exocytotic membrane fusion pathways (Jahn and Scheller 2006). Over the past two decades, in-vitro liposome fusion has been proven to be a powerful tool to reconstruct physiological
SNARE-mediated membrane fusion processes (Liu et al. 2017). The reconstitution of the membrane fusion process not only provides direct evidence of the capability of the cognate
SNARE complex in driving membrane fusion but also allows researchers to study the functional mechanisms of regulatory proteins in related pathways (Wickner and Rizo 2017). Heretofore, a variety of delicate methods for in-vitro
SNARE-mediated liposome fusion have been established (Bao et al. 2018; Diao et al. 2012; Duzgunes 2003; Gong et al. 2015; Heo et al. 2021; Kiessling et al. 2015; Kreye et al. 2008; Kyoung et al. 2013; Liu et al. 2017; Scott et al. 2003). Although technological advances have made reconstitution more physiologically relevant, increasingly elaborate experimental procedures, instruments, and data processing algorithms nevertheless hinder the non-experts from setting up basic SNARE-mediated liposome fusion assays. Here, we describe a low-cost, timesaving, and easy-to-handle protocol to set up a foundational in-vitro
SNARE-mediated liposome fusion assay based on our previous publications (Liu et al. 2023; Wang and Ma 2022). The protocol can be readily adapted to assess various types of
SNARE-mediated membrane fusion and the actions of fusion regulators by using appropriate alternative additives (e.g., proteins, macromolecules, chemicals, etc.). The total time required for one round of the assay is typically two days and could be extremely compressed into one day.