快速实现 SNARE 介导的脂质体融合的实用指南。

Shen Wang, Cong Ma
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引用次数: 0

摘要

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细胞外膜融合途径的引擎(Jahn 和 Scheller,2006 年)。过去二十年来,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有力工具(Liu 等人,2017 年)。膜融合过程的重构不仅为同源 SNARE 复合物驱动膜融合的能力提供了直接证据,还能让研究人员研究相关通路中调控蛋白的功能机制(Wickner 和 Rizo,2017 年)。迄今为止,体外 SNARE 介导的脂质体融合已有多种精细方法(Bao 等人,2018 年;Diao 等人,2012 年;Duzgunes,2003 年;Gong 等人,2015 年;Heo 等人,2021 年;Kiessling 等人,2015 年;Kreye 等人,2008 年;Kyoung 等人,2013 年;Liu 等人,2017 年;Scott 等人,2003 年)。尽管技术的进步使重组更贴近生理,但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业人员建立基本的 SNARE 介导的脂质体融合实验。在此,我们介绍一种低成本、省时且易于操作的方案,以我们之前发表的论文(Liu 等,2023;Wang 和 Ma,2022)为基础,建立体外 SNARE 介导的脂质体融合基础实验。通过使用适当的替代添加剂(如蛋白质、大分子、化学物质等),该方案可以很容易地进行调整,以评估 SNARE 介导的各种类型的膜融合以及融合调节剂的作用。进行一轮测定通常需要两天时间,但可在一天内完成。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A practical guide for fast implementation of SNARE-mediated liposome fusion.

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.

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