Betancourt-Solis Miguel A, McNew James A
Department of BioSciences, Rice University;
Department of BioSciences, Rice University.
J Vis Exp. 2019 Jul 3(149). doi: 10.3791/59867.
Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.
膜融合是真核细胞中的一个关键过程。催化融合需要特定的蛋白质。Atlastin是内质网（ER）驻留蛋白，与内质网的同型融合有关。我们在此详细介绍一种通过两轮亲和层析纯化谷胱甘肽S-转移酶（GST）和多组氨酸标签的果蝇Atlastin的方法。体外研究融合反应需要将纯化的融合蛋白插入脂质双层中。脂质体是理想的模型膜，因为脂质组成和大小可以调节。为此，我们描述了一种通过去除去污剂将果蝇Atlastin重组到预先形成的脂质体中的方法。虽然有几种重组方法可用，但通过去除去污剂进行重组有几个优点，使其适用于Atlastin和其他类似蛋白质。该方法的优点包括高重组产率和重组蛋白的正确取向。该方法可以扩展到其他膜蛋白以及其他需要蛋白脂质体的应用。此外，我们描述了一种基于荧光共振能量转移（FRET）的蛋白脂质体脂质混合测定法，用于测量膜融合。
