Abstract
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.
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Acknowledgements
ZM and KK thank Department of Biotechnology, Government of India, for grand (BT/PR12502/BRB/10/1387/2015) and ZM thanks UGC for fellowship. MSI acknowledges funding from Science and Engineering Research Board grant and JC Bose fellowship. ZM and KK would like to thank Dr. Maurizio Polentarutti of XRD1 beamline at Elettra synchrotron for help in testing LCP crystals.
Funding
DBT Govt. India, Project Number BT/PR12502/BRB/10/1387/2015 and Project Number SERB No: EMR/2016/000641 and JCB/2019/000020 were supported by SERB.
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ZM and KK designed the experiments and ZM performed all the experiments. PD and AP provided all the in-house lipids for screening. MSI provided the Asrij constructs and other reagents. KK supervised overall work. ZM and KK wrote the manuscript with inputs from MSI. Other authors provided editorial inputs.
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Motiwala, Z., Darne, P., Prabhune, A. et al. Expression, Purification and Crystallization of Asrij, A Novel Scaffold Transmembrane Protein. J Membrane Biol 254, 65–74 (2021). https://doi.org/10.1007/s00232-020-00166-7
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DOI: https://doi.org/10.1007/s00232-020-00166-7