The Journal of Membrane Biology ( IF 2.3 ) Pub Date : 2021-01-12 , DOI: 10.1007/s00232-020-00166-7 Zenia Motiwala 1, 2 , Priti Darne 1 , Asmita Prabhune 1 , Maneesha S Inamdar 3 , Kiran Kulkarni 1, 2
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Asrij/OCIAD1 is a scaffold transmembrane protein belonging to the Ovarian Carcinoma Immunoreactive Antigen Domain containing protein family. In Drosophila and mouse models, Asrij localizes at the endosomal and mitochondrial membrane and is shown to regulate the stemness of hematopoietic stem cells. Interaction of Asrij with ADP Ribosylation Factor 1 (Arf1) is shown to be crucial for hematopoietic niche function and prohemocyte maintenance. Here, we report the heterologous expression, standardization of detergents and purification methodologies for crystallization of Asrij/OCIAD1. To probe the activity of bacterially expressed Asrij, we developed a protein complementation assay and conclusively show that Asrij and Arf1 physically interact. Further, we find that sophorolipids improve the solubility and monodispersibility of Asrij. Hence, we propose that sophorolipids could be novel additives for stabilization of membrane proteins. To our knowledge, this is the first study detailing methodology for the production and crystallization of a heterologously expressed scaffold membrane protein and will be widely applicable to understand membrane protein structure and function.
Graphic Abstract
中文翻译：
新型支架跨膜蛋白 Asrij 的表达、纯化和结晶
Asrij/OCIAD1 是一种支架跨膜蛋白，属于含有卵巢癌免疫反应性抗原结构域的蛋白家族。在果蝇在小鼠模型和小鼠模型中，Asrij 定位于内体和线粒体膜，并显示可调节造血干细胞的干性。Asrij 与 ADP 核糖基化因子 1 (Arf1) 的相互作用被证明对造血生态位功能和原血细胞维持至关重要。在这里，我们报告了 Asrij/OCIAD1 结晶的异源表达、去污剂的标准化和纯化方法。为了探测细菌表达的 Asrij 的活性，我们开发了一种蛋白质互补分析，并最终表明 Asrij 和 Arf1 物理相互作用。此外，我们发现槐糖脂提高了 Asrij 的溶解度和单分散性。因此，我们提出槐糖脂可能是稳定膜蛋白的新型添加剂。据我们所知，
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